Alamethicin Topology in Phospholipid Membranes by Oriented Solid-state NMR and EPR Spectroscopies: a Comparison

Abstract

Alamethicin, a hydrophobic peptide that is considered a paradigm for membrane channel formation, was uniformly labeled with 15N, reconstituted into oriented phosphatidylcholine bilayers at concentrations of 1 or 5 mol %, and investigated by solid-state NMR spectroscopy as a function of temperature. Whereas the peptide adopts a transmembrane alignment in POPC bilayers at all temperatures investigated, it switches from a transmembrane to an in-plane orientation in DPPC membranes when passing the phase transition temperature. This behavior can be explained by an increase in membrane hydrophobic thickness and the resulting hydrophobic mismatch condition. Having established the membrane topology of alamethicin at temperatures above and below the phase transition, ESEEM EPR was used to investigate the water accessibility of alamethicin synthetic analogues carrying the electron spin label TOAC residue at one of positions 1, 8, or 16. Whereas in the transmembrane alignment the labels at positions 8 and 16 are screened from the water phase, this is only the case for the latter position when adopting an orientation parallel to the surface. By comparing the EPR and solid-state NMR data of membrane-associated alamethicin it becomes obvious that the TOAC spin labels and the cryo-temperatures required for EPR spectroscopy have less of an effect on the alamethicin-POPC interactions when compared to DPPC. Finally, at P/L ratios of 1/100, spectral line broadening due to spin-spin interactions and thereby peptide oligomerization within the membrane were detected for transmembrane alamethicin.

Alamethicin Supramolecular Organization in Lipid Membranes from 19F Solid-State NMR

“…The data suggest that at room temperature, peptides exhibit a rather dynamic behavior in the membrane (Figs. 4 and 5), and extend the results of previous experiments in which the aggregate size (1,2,16), membrane topology (1,22,23), detailed secondary structure (46,66), and relative alignment of the amphipathic interfaces were shown to be in exchange and regulated by environmental parameters.…”

“…At similar concentrations, previous 15 N solid-state NMR experiments on 15 N-labeled ALM reconstituted into oriented POPC membranes indicated a transmembrane helix orientation (46,55). The high peptide/lipid ratio not only allows for direct comparison with previous NMR studies but also maximizes the 19 F signal intensity.…”

“…In doing so, they adopt a variety of configurations and even change from in-plane to transmembrane orientations as a function of the lipid composition, membrane hydration, peptide/lipid ratio, and transmembrane voltage (1,22,23). In combination with other structural (46,55) and biophysical (1,2) data, the CODEX experiments presented here provide valuable additional information about the oligomerization size of peptides within lipid bilayers, which is difficult to obtain by other methods.…”

“…1. The three Glu(OMe)-for-Gln substitutions do not affect the conformation and functionality of native ALM F50/5 (44)(45)(46)(47).…”

Membrane-Bound Conformations of Antimicrobial Agents and Their Modes of Action

“…In the toroidal-pore model, the AMPs induce the bending of the lipid head groups in a toroidal mode, after binding to them, and form transmembrane pores, and unlike barrel-stave model, the lumen of the pore is lined with both the peptide residues as well as the lipid head groups. AMPs like magainin, melittin, and protegrins have been shown to disrupt the membrane in toroidal mode [62,63], while helical peptides like alamethicin are known to function through barrel-stave mode [64][65][66].…”