Conformation of Alamethicin in Oriented Phospholipid Bilayers Determined by 15N Solid-State Nuclear Magnetic Resonance

Abstract

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

Voltage-dependent energetics of alamethicin monomers in the membrane

“…In the voltage-dependent partitioning model, partitioning of the peptide between the membrane and aqueous phase requires diffusion of the peptide into the membrane. However, our calculations, and experiments [5,33,35,37,38], show that ALM strongly partitions into the membrane regardless of voltage.…”

“…Thus, slight differences in experimental setup might tip the balance towards one or the other. 15N NMR studies showed a tilted orientation of ALM in the bilayer in the nonconductive state [38]. The tilt was between 10°and 20°.…”

Alignment and structural analysis of membrane polypeptides by 15N and 31P solid‐state NMR spectroscopy

“…Similarly, the transmembrane region of the related M2 polypeptide of influenza A has been investigated by solid-state NMR spectroscopy (62 (15,64,65) and the putative channel-lining sequence M2 of the acetyl choline receptor (35). In recent years there has been considerable interest in exploring the utility of natural antibiotic peptides by themselves or as templates to design derivatives for pharmaceutical use (66).…”

Recent Solid-State NMR Studies of Membrane-Bound Peptides and Proteins

“…From the anisotropic 15 N chemicalshift data and 1 H-15 N dipolar couplings determined for alamethicin with 15 N-labeled Ala6, Val9, and Val15 incorporated into a phospholipid bilayer at a peptide-to-lipid molar ratio of 1:8, it was determined that alamethicin assumes a largely linear α-helical structure that spans the membrane with the molecular axis tilted by 10-20°relative to the bilayer normal. In particular, the compatibility with a straight α-helix was tilted by 17°and a slightly kinked MD structure was tilted by 11°relative to the bilayer normal [127]. Measurement of the orientation-dependent 1 H-15 N dipole-dipole coupling, 15 N anisotropic chemical shift, and 2 H quadrupole coupling parameters for a single residue, combined with analysis of the anisotropic interaction for the Aib8 residue, provided detailed information regarding the helix-tilt angle, wobbling, and oscillatory rotation around the helical axis in the membrane-bound state of alamethicin [73].…”