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Melittin is a venom peptide that disrupts lipid bilayers at temperatures below the liquid-crystalline to gel phase transition temperature (T c ). Notably, the ability of melittin to disrupt acidic dimyristoylphosphatidylglycerol (DMPG) bilayers was weaker than its ability to disrupt neutral dimyristoylphosphatidylcholine bilayers. The structure and orientation of melittin bound to DMPG bilayers were revealed by analyzing the 13 C chemical shift anisotropy of [1- 13 C]-labeled melittin obtained from solid-state 13 C NMR spectra. 13 C chemical shift anisotropy showed oscillatory shifts with the index number of residues. Analysis of the chemical shift oscillation properties indicated that melittin bound to a DMPG membrane adopts a bent α-helical structure with tilt angles for the N- and C-terminal helices of -32 and +30°, respectively. The transmembrane melittin in DMPG bilayers indicates that the peptide protrudes toward the C-terminal direction from the core region of the lipid bilayer to show a pseudotransmembrane bent α-helix. Molecular dynamics simulation was performed to characterize the structure and interaction of melittin with lipid molecules in DMPG bilayers. The simulation results indicate that basic amino acid residues in melittin interact strongly with lipid head groups to generate a pseudo-transmembrane alignment. The N-terminus is located within the lipid core region and disturbs the lower surface of the lipid bilayer.

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Bombolitin II (BLT2) is one of the hemolytic heptadecapeptides originally isolated from the venom of a bumblebee. Structure and orientation of BLT2 bound to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) membranes were determined by solid-state (31)P and (13)C NMR spectroscopy. (31)P NMR spectra showed that BLT2-DPPC membranes were disrupted into small particles below the gel-to-liquid crystalline phase transition temperature (T(c)) and fused to form a magnetically oriented vesicle system where the membrane surface is parallel to the magnetic fields above the T(c). (13)C NMR spectra of site-specifically (13)C-labeled BLT2 at the carbonyl carbons were observed and the chemical shift anisotropies were analyzed to determine the dynamic structure of BLT2 bound to the magnetically oriented vesicle system. It was revealed that the membrane-bound BLT2 adopted an α-helical structure, rotating around the membrane normal with the tilt angle of the helical axis at 33°. Interatomic distances obtained from rotational-echo double-resonance experiments further showed that BLT2 adopted a straight α-helical structure. Molecular dynamics simulation performed in the BLT2-DPPC membrane system showed that the BLT2 formed a straight α-helix and that the C-terminus was inserted into the membrane. The α-helical axis is tilted 30° to the membrane normal, which is almost the same as the value obtained from solid-state NMR. These results suggest that the membrane disruption induced by BLT2 is attributed to insertion of BLT2 into the lipid bilayers., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)