Conformation and Dynamics of Melittin Bound to Magnetically Oriented Lipid Bilayers by Solid-State 31P and 13C NMR Spectroscopy

Abstract

The conformation and dynamics of melittin bound to the dimyristoylphosphatidylcholine (DMPC) bilayer and the magnetic orientation in the lipid bilayer systems were investigated by solid-state (31)P and (13)C NMR spectroscopy. Using (31)P NMR, it was found that melittin-lipid bilayers form magnetically oriented elongated vesicles with the long axis parallel to the magnetic field above the liquid crystalline-gel phase transition temperature (T(m) = 24 degrees C). The conformation, orientation, and dynamics of melittin bound to the membrane were further determined by using this magnetically oriented lipid bilayer system. For this purpose, the (13)C NMR spectra of site-specifically (13)C-labeled melittin bound to the membrane in the static, fast magic angle spinning (MAS) and slow MAS conditions were measured. Subsequently, we analyzed the (13)C chemical shift tensors of carbonyl carbons in the peptide backbone under the conditions where they form an alpha-helix and reorient rapidly about the average helical axis. Finally, it was found that melittin adopts a transmembrane alpha-helix whose average axis is parallel to the bilayer normal. The kink angle between the N- and C-terminal helical rods of melittin in the lipid bilayer is approximately 140 degrees or approximately 160 degrees, which is larger than the value of 120 degrees determined by x-ray diffraction studies. Pore formation was clearly observed below the T(m) in the initial stage of lysis by microscope. This is considered to be caused by the association of melittin molecules in the lipid bilayer.

Solid‐state NMR analysis of the orientation and dynamics of epigallocatechin gallate, a green tea polyphenol, incorporated into lipid bilayers

“…Since the surface of the bicelle is parallel to the magnetic field, [4-2 H]EGCg molecules rotate about the bilayer normal with a constant tilt angle to the axis. Similar dynamic pictures have been observed for other membrane-bound molecules such as melittin (transmembrane type) [20] and dynorphin (surface type). [27] Furthermore, the intensity of the central lines increased, and the splitting of the orientated spectra decreased as the pulse interval increased.…”

Mechanism of Lipid Bilayer Disruption by the Human Antimicrobial Peptide, LL-37

“…In the 31 P spectra of oriented lipid bilayers with LL-37 incorporated, no isotropic peak characteristic small bilayer fragments such as micelles or small disks was observed (Figures 8 and 9). Isotropic peaks are clearly present in 31 P spectra of both oriented and unoriented bilayers perturbed by other peptides which are known to induce micellization of the membrane, such as melittin (36), mastoparan (35), nisin (52), or δ-lysin (39). In the case of protegrin-1 (53), an isotropic peak was not observed, but significant averaging of the 31 P CSA did occur, and was attributed to the formation of small disklike fragments from the membrane.…”

NMR methods for studying membrane‐active antimicrobial peptides

“…As outlined above, these chemical shifts depend on the secondary structure and have been used to discriminate ␣-helical and ␤-strand conformations (89 -91). When several positions are labeled, the orientation of the helix can be determined from the anisotropic "chemical shift wave" obtained from oriented samples (86,90). The chemical shift has also been studied as a function of pH to reveal the effect of side chain protonation (92).…”