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Mechanisms of peptide-induced pore formation in lipid bilayers investigated by oriented 31P solid-state NMR spectroscopy.

Bertelsen K, Dorosz J, Hansen SK, Nielsen NC, Vosegaard T - PLoS ONE (2012)

Bottom Line: For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring.These lipids display reduced dynamics.Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

View Article: PubMed Central - PubMed

Affiliation: Center for Insoluble Protein Structures (inSPIN), Department of Chemistry, University of Aarhus, Aarhus, Denmark.

ABSTRACT
There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP's interaction with lipid membranes. In this study we show that oriented-sample (31)P solid-state NMR spectroscopy can be used to probe the membrane perturbations and disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

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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.
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pone-0047745-g005: 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.

Mentions: A number of samples were prepared to study the interaction between novicidin and lipid bilayers. 31P oriented solid-state NMR spectra for these samples are shown in Figure 5. In all samples, the lipid bilayer consist of a mixture of DMPC:DMPG in 80∶20 molar ratio. The pure-lipid sample (Fig. 5a) displays a single sharp resonance at 28 ppm. This indicates that the lipids in the bilayer are oriented along the magnetic field axis. However, if the alignment were perfect, we would expect the peak to show up exactly at the frequency of the left edge of the powder spectrum for the DMPC vesicles (31 ppm) as observed for the pure DMPC sample (Fig. 4a). The lower resonance frequency observed here may suggests that the mixed-lipid sample has a lower order parameter of . In an alternative interpretation, we can use the thinned-bilayer model to explain the lower resonance frequency, providing a more detailed description of the system. We note that the slight disorder of the lipid is highly dynamic since the observed resonance is narrow and symmetric. Static disorder (mosaic spread) would introduce significant line broadening and an asymmetric line shape extending towards the isotropic chemical shift [37]. A fit of the spectrum (Fig. 5b) using the thinned-bilayer model shows that the peak at ∼28 ppm corresponds to lipid molecules diffusing at a rate of approximately 10−8 cm2/s in bilayers thinned by 1.5 Å as shown in Table 2. These thinning effects may be caused by the mixture of the charged and zwitterionic lipids.


Mechanisms of peptide-induced pore formation in lipid bilayers investigated by oriented 31P solid-state NMR spectroscopy.

Bertelsen K, Dorosz J, Hansen SK, Nielsen NC, Vosegaard T - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475706&req=5

pone-0047745-g005: 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.
Mentions: A number of samples were prepared to study the interaction between novicidin and lipid bilayers. 31P oriented solid-state NMR spectra for these samples are shown in Figure 5. In all samples, the lipid bilayer consist of a mixture of DMPC:DMPG in 80∶20 molar ratio. The pure-lipid sample (Fig. 5a) displays a single sharp resonance at 28 ppm. This indicates that the lipids in the bilayer are oriented along the magnetic field axis. However, if the alignment were perfect, we would expect the peak to show up exactly at the frequency of the left edge of the powder spectrum for the DMPC vesicles (31 ppm) as observed for the pure DMPC sample (Fig. 4a). The lower resonance frequency observed here may suggests that the mixed-lipid sample has a lower order parameter of . In an alternative interpretation, we can use the thinned-bilayer model to explain the lower resonance frequency, providing a more detailed description of the system. We note that the slight disorder of the lipid is highly dynamic since the observed resonance is narrow and symmetric. Static disorder (mosaic spread) would introduce significant line broadening and an asymmetric line shape extending towards the isotropic chemical shift [37]. A fit of the spectrum (Fig. 5b) using the thinned-bilayer model shows that the peak at ∼28 ppm corresponds to lipid molecules diffusing at a rate of approximately 10−8 cm2/s in bilayers thinned by 1.5 Å as shown in Table 2. These thinning effects may be caused by the mixture of the charged and zwitterionic lipids.

Bottom Line: For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring.These lipids display reduced dynamics.Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

View Article: PubMed Central - PubMed

Affiliation: Center for Insoluble Protein Structures (inSPIN), Department of Chemistry, University of Aarhus, Aarhus, Denmark.

ABSTRACT
There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP's interaction with lipid membranes. In this study we show that oriented-sample (31)P solid-state NMR spectroscopy can be used to probe the membrane perturbations and disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

Show MeSH