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

Abstract

Alamethicins (ALMs) are antimicrobial peptides of fungal origin. Their sequences are rich in hydrophobic amino acids and strongly interact with lipid membranes, where they cause a well-defined increase in conductivity. Therefore, the peptides are thought to form transmembrane helical bundles in which the more hydrophilic residues line a water-filled pore. Whereas the peptide has been well characterized in terms of secondary structure, membrane topology, and interactions, much fewer data are available regarding the quaternary arrangement of the helices within lipid bilayers. A new, to our knowledge, fluorine-labeled ALM derivative was prepared and characterized when reconstituted into phospholipid bilayers. As a part of these studies, C 19 F 3 -labeled compounds were characterized and calibrated for the first time, to our knowledge, for 19 F solid-state NMR distance and oligomerization measurements by centerband-only detection of exchange (CODEX) experiments, which opens up a large range of potential labeling schemes. The 19 F-19 F CODEX solid-state NMR experiments performed with ALM in POPC lipid bilayers and at peptide/lipid ratios of 1:13 are in excellent agreement with molecular-dynamics calculations of dynamic pentameric assemblies. When the peptide/lipid ratio was lowered to 1:30, ALM was found in the dimeric form, indicating that the supramolecular organization is tuned by equilibria that can be shifted by changes in environmental conditions.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers

“…A plateau of for S / S 0 can also be obtained from linear combinations of other oligomeric structures (see analysis of data obtained earlier on for alamethicin, in the Supporting Information of ref. ). For example, a combination of 10 % monomer and 90 % hexamer could also result in a plateau of 0.25.…”

“…Notably, for a predominantly tetrameric arrangement the statistical probability to also have inter‐oligomeric contacts between tetramers affecting the CODEX decay curve is rather small and can be ignored (see the Supporting Information of ref. ). The exact distance between monomers is not only dependent on the statistical errors (as indicated by the bars in Figure ) but also on the accuracy of the determination of F (0); this makes the calibration shown in Figure so important.…”

“…To this end, [ 19 F‐Phe10]trichogin was reconstituted into POPC bilayers and studied by 19 F MAS solid‐state NMR spectroscopy. The 19 F CODEX experiments allow one to count the 19 F spins that are interacting within a radius of 15 Å, to measure the dipolar interactions and thereby the distances between these nuclei . The experiment drives the exchange of magnetization due to dipolar interactions—the signal under mixing conditions S relative to the corresponding signal without exchange S 0 is recorded as a function of the mixing time.…”

“…19 F‐CODEX experiments were performed as described in refs. and . The normalized intensity, S / S 0 , was measured as a function of the mixing time until it reached a plateau.…”

“…Under fast magic‐angle spinning (MAS), the experiment was initially designed to reveal the correlation times and motional regimes at ambient temperatures . However, when such motions are frozen at low temperatures, the CODEX technique allows one to elucidate the aggregated structure through distances in the range of up to 15 Å between specifically 19 F‐labeled residues . Furthermore, the experiment reveals the number of subunits in the aggregate structure.…”