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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 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.
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pone-0047745-g003: 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.

Mentions: Before turning to the 31P investigations of the two peptide-lipid complexes, we investigated the pore formation of alamethicin and novicidin at high P:L ratios. At high P:L ratios, oriented-sample 15N solid-state NMR is an ideal technique to probe the membrane-anchored conformation of the peptides [16]. We prepared samples of 15N-Aib8 alamethicin and 15N-Ile14 novicidin at high peptide-to-lipid (P:L) ratios (∼1∶15) and recorded the oriented-sample 15N solid-state NMR spectra shown in Fig. 3a and 3b. These spectra display a clear signature of oriented samples, i.e., the spectra show narrow peaks and no powder patterns, although the resonance for alamethicin is somewhat broad due to the inhomogeneous nature of the alamethicin pores and mosaic spread [37], [39], [56].


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 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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475706&req=5

pone-0047745-g003: 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.
Mentions: Before turning to the 31P investigations of the two peptide-lipid complexes, we investigated the pore formation of alamethicin and novicidin at high P:L ratios. At high P:L ratios, oriented-sample 15N solid-state NMR is an ideal technique to probe the membrane-anchored conformation of the peptides [16]. We prepared samples of 15N-Aib8 alamethicin and 15N-Ile14 novicidin at high peptide-to-lipid (P:L) ratios (∼1∶15) and recorded the oriented-sample 15N solid-state NMR spectra shown in Fig. 3a and 3b. These spectra display a clear signature of oriented samples, i.e., the spectra show narrow peaks and no powder patterns, although the resonance for alamethicin is somewhat broad due to the inhomogeneous nature of the alamethicin pores and mosaic spread [37], [39], [56].

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