Structure and alignment of the membrane-associated peptaibols ampullosporin a and alamethicin by oriented15N and31P 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 α-tetrasubstituted amino acid residue α-amino/sobutyric acid. Here ampullosporin A and alamethicin were uniformly labeledb with 15N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled 15N and 31P 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 15N chemical shift - 1H-15N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed α-/310-helical structures which can be explained by the restraints imposed by the membranes and the bulky α-amino/sobutyric acid residues. The 15N 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. © 2009 by the Biophysical Society.