Structure and Alignment of the Membrane-Associated Peptaibols Ampullosporin A and Alamethicin by Oriented 15N and 31P Solid-State NMR Spectroscopy

Abstract

Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the alpha-tetrasubstituted amino acid residue ?-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with (15)N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled (15)N and (31)P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional (15)N chemical shift -(1)H-(15)N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed alpha-/3(10)-helical structures which can be explained by the restraints imposed by the membranes and the bulky alpha-aminoisobutyric acid residues. The (15)N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides.

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

“…Notably, the tilt angles of the helices with respect to the membrane normal of the structures shown in Fig. 4 d agree well with those obtained by oriented solid-state NMR of 15 N-labeled ALM in POPC membranes (55).…”

“…4 c) and the structures obtained from MD calculations (Fig. 4 d) agree in that considerable heterogeneity characterizes the arrangement of helices and side chains (16,55,60). Notably, the corresponding conformational fluctuations have been analyzed in terms of conductivities through the resulting pore (61).…”

“…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.…”

Membrane structure and conformational changes of the antibiotic heterodimeric peptide distinctin by solid-state NMR spectroscopy

“…S1 C-E). Whereas hydrophobic mismatch-dependent topological alterations (19,20) or interactions between transmembrane helical peptides have been studied previously in considerably detail (21)(22)(23), the data shown here provide a unique example where helix-helix interactions are described at an interfacial bilayer location. A significant increase in helix conformations has also been observed when the concentration of linear amphipathic antimicrobial peptides in aqueous buffer is increased or their solubility reduced by, for example, a change in pH (24)(25)(26).…”