Kazushi Norisada

Conformation and dynamics of melittin bound to magnetically oriented lipid bilayers by solid-state (31)P and (13)C NMR spectroscopy.

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.

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

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 Structure and Orientation of Melittin Bound to Acidic Lipid Bilayers, As Revealed by Solid-State NMR and Molecular Dynamics Simulation

Melittin is a venom peptide that disrupts lipid bilayers at temperatures below the liquid-crystalline to gel phase transition temperature (Tc). 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 13C chemical shift anisotropy of [1-13C]-labeled melittin obtained from solid-state 13C NMR spectra. 13C 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.

Structure Determination of Membrane Peptides and Proteins by Solid-State NMR

Solid-state nuclear magnetic resonance (NMR) spectroscopy provides useful information on the structure, topology, and orientation of peptides and proteins bound to lipid bilayers. The structure and orientation of membrane-associated peptides and proteins can be elucidated by analyzing structural constraints obtained from anisotropic chemical-shift interactions, nuclear dipolar interactions, or a combination of these interactions. Detailed structures of various peptides and proteins in their membrane-bound states can be studied by analyzing anisotropic chemical-shift interactions by, for example, chemical-shift oscillation analysis, and nuclear dipolar interactions using techniques such as polarity index slant angle wheel analysis. Magic-angle spinning (MAS) experiments coupled with cross-polarization (CP) and high-power decoupling (CP-MAS) techniques provide high-resolution 13C and 15N NMR signals for selectively or uniformly labeled membrane-bound peptides and proteins in solid-state NMR. Furthermore, homonuclear and heteronuclear dipolar interactions can be recoupled using various spin manipulation pulse sequences under MAS conditions. These experiments enable the correlation of 13C–13C and 13C–15N signals, allowing their assignment to specific amino acid residues and ultimately determination of the high-resolution structure of membrane-bound peptides and proteins.