The membrane-active, cationic, β-hairpin peptide, arenicin, isolated from marine polychaeta Arenicola marina exhibits a broad spectrum of antimicrobial activity. The peptide in aqueous solution adopts the significantly twisted β-hairpin conformation without pronounced amphipathicity. To assess the mechanism of arenicin action, the spatial structure and backbone dynamics of the peptide in membrane-mimicking media and its pore-forming activity in planar lipid bilayers were studied. The spatial structure of the asymmetric arenicin dimer stabilized by parallel association of N-terminal strands of two β-hairpins was determined using triple-resonance nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles. Interaction of arenicin with micelles and its oligomerization significantly decreased the right-handed twist of the β-hairpin, increased its amphipathicity, and led to stabilization of the peptide backbone on a picosecond to nanosecond time scale. Relaxation enhancement induced by water-soluble (Mn(2+)) and lipid-soluble (16-doxylstearate) paramagnetic probes pointed to the dimer transmembrane arrangement. Qualitative NMR and circular dichroism study of arenicin-2 in mixed DPC/1,2-dioleoyl-sn-glycero-3-phosphoglycerol bicelles, sodium dodecyl sulfate micelles, and lipid vesicles confirmed that a similar dimeric assembly of the peptide was retained in membrane-mimicking systems containing negatively charged lipids and detergents. Arenicin-induced conductance was dependent on the lipid composition of the membrane. Arenicin low-conductivity pores were detected in the phosphatidylethanolamine-containing lipid mixture, whereas the high-conductivity pores were observed in an exclusively anionic lipid system. The measured conductivity levels agreed with the model in which arenicin antimicrobial activity was mediated by the formation of toroidal pores assembled of two, three, or four β-structural peptide dimers and lipid molecules. The structural transitions involved in arenicin membrane-disruptive action are discussed.

The structure of the channel-forming polypeptide gramicidin A (GA) incorporated into phosphatidyl-choline (PC) liposomes has been studied as a function of the degree of unsaturation of the acyl chains of PC. The initial conformational state of GA in reconstituted bilayers is determined by the solvent in which the peptide and the lipid are initially co-dissolved, whereas the equilibrium conformational state (after heat incubation) is affected by the lipid structure rather than by the nature of the solvent. The conformational equilibrium of GA has been studied in liposomes prepared from PC having a variable number of double bonds in the fatty acid moiety, by circular dichroism and Fourier transform infrared. Liposomes were prepared from trifluoroethanol or ethanol solutions and incubated at 68 degrees C. GA was shown to retain the conformation of the right-handed pi-->6.3 pi<--6.3 helix in PC with saturated acyl chains and with one double bond, whereas in dilinoleoyl-PC, having two double bond in each chain, the thermodynamically preferred structures are left-handed antiparallel and parallel double pi pi 5.6 helices. Natural soybean PC also favours left-handed pi pi 5.6 helical structures of GA (approximately 75%). This finding is discussed in terms of the role of PC unsaturation in the dynamic properties of the lipid matrix. Differences between observed FTIR spectra of the increases decreases pi pi 5.6 helix in solution (and to a larger extent in the membrane) and the calculated IR spectra can be interpreted as resulting from deviation of the real structure from the theoretically derived ideal helix. The data obtained provide grounds for better understanding of a GA channel functioning in lipids of variable degrees of unsaturation.

The formation of spherical and monodisperse small unilamellar vesicles is observed upon temperature jumps in a mixed system composed of a phospholipid (dimyristoylphosphatidylcholine) and a bile salt (sodium cholate) in an aqueous buffer. In order to enhance the X-ray contrast of the system a mixed water/sucrose buffer is used. The spontaneous formation of unilamellar structures from mixed micelles upon temperature increase and the reverse solubilisation of membranes upon cooling is studied by means of synchrotron radiation small-angle X-ray scattering. The variations of the vesicle size are presented when the temperature and the surfactant / lipid ratio are modified. The kinetic conditions leading to monodisperse vesicles as well as the reversibility of the micelle to vesicle transition are investigated.

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The transition states of binary mixtures of dipalmitoyl-and dimyristoylphosphatidylcholines with sodium cholate at the reversible temperature-induced micellar-lamellar transformation were characterized by turbidimetry, electron microscopy, 31P NMR and differential scanning calorimetry. This transformation is triggered by the phospholipid acyl chain melting, and appears to include two structural pathways: (i) from discoidal mixed micelles to network-like structures composed of long interlaced rod-like micelles, then to multilayer membrane structures, and finally to multilamellar vesicles; and (ii) from discoidal micelles to membrane fragments and finally to unilamellar vesiCleS.