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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 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.
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pone-0047745-g004: 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.

Mentions: Having established reasonable models for the high-P:L peptide anchoring in consistency with previously suggested models, we will now investigate both the low-P:L and high-P:L regions by 31P oriented solid-state NMR to establish more insight into the membrane anchoring mechanisms of the peptides. To study these features for alamethicin, several samples containing mixtures of alamethicin and lipids with different P:L molar ratios were prepared under careful attention to the procedures leading to the reference high-P:L samples. The 31P solid-state NMR spectra for these oriented samples are shown in Figure 4 along with simulations employing the above diffusion model with the parameters listed in Table 1. For the different samples, only the amount of alamethicin was changed but all contained the same amount of lipids, thereby varying the P:L ratio from 1∶400 to 1∶25. The peaks at 15–30 ppm may be simulated using the thinned bilayer model, while the peaks at −16 ppm attributed to lipids with a perpendicular orientation are represented by Gaussian lines. The use of thinned bilayer geometry is rationalized by assuming that regions of the lipid bilayer distant to peptides or with surface-bound peptide can be modeled by the thinned bilayer model (Fig. 2a) with a normal diffusion rate (10−8 cm2/s) [28], [60] while regions of the lipid in closer contact with the peptides also display thinning but have reduced dynamics (10−10 cm2/s). We note that the dependence on the diffusion rate is not as pronounced as the dependence on the geometrical parameters defined in Figure 2, so the diffusion rates of 10−8 cm2/s and 10−10 cm2/s should not be taken as absolute values, but represent the order of magnitude of the diffusion rates. In addition to the spectral components discussed above, it is relevant to investigate whether the individual samples contain any unoriented components, which would be manifested as a vesicle powder pattern (cf. Figure 1c). Hence this component is included in all simulations.


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 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475706&req=5

pone-0047745-g004: 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.
Mentions: Having established reasonable models for the high-P:L peptide anchoring in consistency with previously suggested models, we will now investigate both the low-P:L and high-P:L regions by 31P oriented solid-state NMR to establish more insight into the membrane anchoring mechanisms of the peptides. To study these features for alamethicin, several samples containing mixtures of alamethicin and lipids with different P:L molar ratios were prepared under careful attention to the procedures leading to the reference high-P:L samples. The 31P solid-state NMR spectra for these oriented samples are shown in Figure 4 along with simulations employing the above diffusion model with the parameters listed in Table 1. For the different samples, only the amount of alamethicin was changed but all contained the same amount of lipids, thereby varying the P:L ratio from 1∶400 to 1∶25. The peaks at 15–30 ppm may be simulated using the thinned bilayer model, while the peaks at −16 ppm attributed to lipids with a perpendicular orientation are represented by Gaussian lines. The use of thinned bilayer geometry is rationalized by assuming that regions of the lipid bilayer distant to peptides or with surface-bound peptide can be modeled by the thinned bilayer model (Fig. 2a) with a normal diffusion rate (10−8 cm2/s) [28], [60] while regions of the lipid in closer contact with the peptides also display thinning but have reduced dynamics (10−10 cm2/s). We note that the dependence on the diffusion rate is not as pronounced as the dependence on the geometrical parameters defined in Figure 2, so the diffusion rates of 10−8 cm2/s and 10−10 cm2/s should not be taken as absolute values, but represent the order of magnitude of the diffusion rates. In addition to the spectral components discussed above, it is relevant to investigate whether the individual samples contain any unoriented components, which would be manifested as a vesicle powder pattern (cf. Figure 1c). Hence this component is included in all simulations.

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