Buforin 2 is an antimicrobial peptide discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans. The 21-residue peptide with +6 net charge shows antimicrobial activity an order of magnitude higher than that of magainin 2, a membrane-permeabilizing antimicrobial peptide from Xenopus laeVis [

The antimicrobial peptide magainin 2 isolated from the skin of the African clawed frog Xenopus laevis crosses lipid bilayers by transiently forming a peptide-lipid supramolecular complex pore inducing membrane permeabilization and flip-flop of membrane lipids [Matsuzaki, K., Murase, O., Fujii, N., and Miyajima, K. (1996) Biochemistry 35, 11361-11368]. In contrast, the antimicrobial peptide buforin 2 discovered in the stomach tissue of the Asian toad Bufo bufo gargarizans efficiently crosses lipid bilayers without inducing severe membrane permeabilization or lipid flip-flop, and the Pro(11) residue plays a key role in this unique property [Kobayashi, S, Takeshima, K., Park, C. B., Kim, S. C., and Matsuzaki, K. (2000) Biochemistry 39, 8648-8654]. To elucidate the translocation mechanism, the secondary structure and the orientation of the peptide in lipid bilayers as well as the effects of the peptide concentration, the lipid composition, and the cis-trans isomerization of the Pro peptide bond on translocation efficiency were investigated. The translocation efficiencies of F10W-buforin 2 (BF2), P11A-BF2, and F5W-magainin 2 (MG2) across egg yolk L-alpha-phosphatidyl-DL-glycerol (EYPG)/egg yolk L-alpha-phosphatidylcholine (1/1) bilayers were dependent supralinearly on the peptide concentration, suggesting that the translocation mechanisms of these peptides are similar. The incorporation of the negative curvature-inducing lipid egg yolk L-alpha -phosphatidylethanolamine completely suppressed the translocation of BF2, indicating the induction of the positive curvature by BF2 on the membrane is related to the translocation process, similarly to MG2. In pure EYPG, where the repulsion between polycationic BF2 molecules is reduced, membrane permeabilization and coupling lipid flip-flop were clearly observed. Structural studies by use of Fourier transform infrared-polarized attenuated total reflection spectroscopy indicated that BF2 assumed distorted helical structures in EYPG/EYPC bilayers. A BF2 analogue with an alpha-methylproline, which fixed the peptide bond to the trans configuration, translocated similarly to the parent peptide, suggesting the cis-trans isomerization of the Pro peptide bond is not involved in the translocation process. These results indicate that BF2 crosses lipid bilayers via a mechanism similar to that of MG2. The presence of Pro(11) distorts the helix, concentrating basic amino acid residues in a limited amphipathic region, thus destabilizing the pore by enhanced electrostatic repulsion, enabling efficient translocation.

Liposomes have frequently been used as models of biomembranes or vehicles for drug delivery. However, the systematic characterization of lipid vesicles by right angle light scattering and turbidity has not been carried out despite the usefulness of such studies for size estimation. In this study, liposomes of various sizes were prepared by sonication and extrusion. The mean cumulant radii of the vesicles were determined by dynamic light scattering. The lamellarities were estimated based on fluorescence quenching of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)dipalmitoyl-L-alpha-phosph ati dylethanolamine by sodium dithionite. Right angle light scattering intensity and optical density at 436 nm per unit lipid concentration were measured as a function of vesicle radius. With a vesicle radius < or =100 nm, the optical parameters could be well explained by the Rayleigh-Gans-Debye theory in which the liposomes were modeled as homogeneous spheres with mean refractive indices determined by the volume fractions of lipids in vesicles.

Investigation of interactions between hydrophobic model peptides and lipid bilayers is perhaps the only way to elucidate the principles of the folding and stability of membrane proteins (White, S. H., and Wimley, W. C. (1998) Biochim. Biophys. Acta 1367, 339-352). We designed the completely hydrophobic "inert" peptide modeling a transmembrane (TM) helix without any of the specific side-chain interactions expected, X-(LALAAAA)(3)-NH(2) [X = Ac (I), 7-nitro-2-1,3-benzoxadiazol-4-yl (II), or 5(6)-carboxytetramethylrhodamine (III)]. Fourier transform infrared-polarized attenuated total reflection measurements revealed that I as well as II assume a TM helix in hydrated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. Dithionite quenching experiments detected no topological change (flip-flop) in the helix II for at least 24 h. Thus, the TM helix itself is a highly stable structure, even in the absence of flanking hydrophilic or aromatic amino acids which are suggested to play important roles in stable TM positioning. Helix self-association in lipid bilayers was detected by fluorescence resonance energy transfer between II and III. The peptide was in a monomer-antiparallel dimer equilibrium with an association free energy of approximately -13 kJ/mol. Electron spin resonance spectra of 1-palmitoyl-2-stearoyl-(14-doxyl)-sn-glycero-3-phosphocholine demonstrated the presence of a motionally restricted component at lower temperatures.