Structure and orientation of antibiotic peptide alamethicin in phospholipid bilayers as revealed by chemical shift oscillation analysis of solid state nuclear magnetic resonance and molecular dynamics simulation

Abstract

The structure, topology and orientation of membrane-bound antibiotic alamethicin were studied using solid state nuclear magnetic resonance (NMR) spectroscopy. (13)C chemical shift interaction was observed in [1-(13)C]-labeled alamethicin. The isotropic chemical shift values indicated that alamethicin forms a helical structure in the entire region. The chemical shift anisotropy of the carbonyl carbon of isotopically labeled alamethicin was also analyzed with the assumption that alamethicin molecules rotate rapidly about the bilayer normal of the phospholipid bilayers. It is considered that the adjacent peptide planes form an angle of 100° or 120° when it forms α-helix or 310-helix, respectively. These properties lead to an oscillation of the chemical shift anisotropy with respect to the phase angle of the peptide plane. Anisotropic data were acquired for the 4 and 7 sites of the N- and C-termini, respectively. The results indicated that the helical axes for the N- and C-termini were tilted 17° and 32° to the bilayer normal, respectively. The chemical shift oscillation curves indicate that the N- and C-termini form the α-helix and 310-helix, respectively. The C-terminal 310-helix of alamethicin in the bilayer was experimentally observed and the unique bending structure of alamethicin was further confirmed by measuring the internuclear distances of [1-(13)C] and [(15)N] doubly-labeled alamethicin. Molecular dynamics simulation of alamethicin embedded into dimyristoyl phophatidylcholine (DMPC) bilayers indicates that the helical axes for α-helical N- and 310-helical C-termini are tilted 12° and 32° to the bilayer normal, respectively, which is in good agreement with the solid state NMR results.

Dynamic membrane interactions of antibacterial and antifungal biomolecules, and amyloid peptides, revealed by solid-state NMR spectroscopy

“…Solid-state NMR is particularly well suited for elucidating the dynamics, topologies, orientations, and high resolution structures of peptides in the lipid bilayer environment using membrane mimetics such as lipid vesicles, bicelles or lipid nanodiscs/macrodiscs. While several studies have reported valuable structural and functional insights into a variety of AMPs (including magainins, MSI-78, LL-37, pardaxin, protegrin and other designed peptides), the use of chemical shift oscillation analysis in elucidating the detailed structure and orientation of the membrane bound state of antibacterial peptides such as melittin [15,20,21], bonbolitin II [49], lactoferrampin [45] and alamethicin [50] reported in the literature are highlighted below.…”

“…Further detailed structural and orientational analyses of membrane-bound alamethicin have been carried out using solid-state NMR spectroscopy by analyzing the chemical shift oscillation patterns [12,50]. 13 C chemical-shift interactions were observed in [1- 13 C]-labeled alamethicin.…”

“…It was, therefore, determined that the N- and C-termini form α- and 3 10 -helices, respectively (Fig. 8) [12,50]. …”

“…8C and D, based on the previously obtained structure by MD simulation [74]. The membrane bound structure of alamethicin determined by solid-state NMR analysis is used as components of a hexagonal alamethicin oligomer [50]. It is of interest to note that Gln7 and Glnl8 residues face to the inside of the ion channel, and thus the inside of the helical bundle shows a hydrophilic character suggesting the high ion conductivity inside the channel.…”

“…The dihedral angles of the peptide planes between the n− and n + 1-residues of the N- and C-termini were determined to be α- and 3 10 -helices, respectively. (B): Structure and topology of alamethicin bound to a DMPC bilayer, as determined from chemical shift oscillation data [50]. Side view (C) and top view (D) of the hexameric oligomer of alamethicin in the membrane environment.…”