Extensive Bilayer Perforation Coupled with the Phase Transition Region of an Anionic Phospholipid

At low ionic strength dimyristoylphosphatidylglycerol (DMPG) exhibits a broad phase transition region characterized by several superimposed calorimetric peaks. Peculiar properties, such as sample transparency, are observed only in the transition region. In this work we use differential scanning calorimetry (DSC), turbidity. and optical microscopy to study the narrowing of the transition region with the increase of ionic strength (0-500 mM NaCl). Upon addition of salt, the temperature extension of the transition region is reduced, and the number of calorimetric peaks decreases until a single cooperative event at T(m) = 23 degrees C is observed in the presence of 500 mM NaCl. The transition region is always coupled with a decrease in turbidity, but a transparent region is detected within the melting process only in the presence of up to 20 mM NaCl. The vanishing of the transparent region is associated with one of the calorimetric peaks. Optical microscopy of giant vesicles shows that bilayers first rupture when the transition region is reached and Subsequently lose optical contrast. Fluorescence microscopy reveals a blurry and undefined image in the transparent region, suggesting a different lipid self-assembly. Overall sample turbidity can be directly related to the bilayer optical contrast. Our observations are discussed in terms of the bilayer being perforated along the transition region. In the narrower temperature interval of the transparent region, dependent on the ionic strength, the perforation is extensive and the bilayer completely loses the optical contrast.

Solution structure and interaction of cupiennin 1a, a spider venom peptide, with phospholipid bilayers

The solution structure of cupiennin 1a, a 35 residue, basic antibacterial peptide isolated from the venom of the spider Cupiennius salei, has been determined by nuclear magnetic resonance (NMR) spectroscopy. The peptide was found to adopt a helix−hinge−helix structure in a membrane mimicking solvent. The hinge may play a role in allowing the amphipathic N-terminal helix and polar C-terminal helix to orient independently upon membrane binding, in order to achieve maximal antibacterial efficacy. Solid-state 31P and 2H NMR was used to further study the effects of cupiennin 1a on the dynamic properties of lipid membranes, using zwitterionic chain deuterated dimyristoylphosphatidylcholine (d54-DMPC) and anionic dimyristoylphosphatidylglycerol (DMPG) multilamellar vesicles. In d54-DMPC alone, cupiennin 1a caused a decrease in the 31P chemical shift anisotropy, indicating some interaction with the lipid head groups, and a decrease in order over the entire acyl chain. In contrast, for the mixed (d54-DMPC/DMPG) lipid system cupiennin 1a appeared to induce lateral separation of the two lipids as evidenced by the 31P spectra, in which the peptide preferentially interacted with DMPG. Little effect was observed on the deuterated acyl chain order parameters in the d54-DMPC/DMPG model membranes. Furthermore, 31P NMR relaxation measurements confirmed a differential effect on the lipid motions depending upon the membrane composition. Therefore, subtle differences are likely in the mechanism by which cupiennin 1a causes membrane lysis in either prokaryotic or eukaryotic cells, and may explain the specific spectrum of activity.

Peptide-induced coalescence of bicellar lipid assemblies: sensitivity to anionic lipid and peptide properties

Bicellar lipid mixture dispersions progressively coalesce to larger structures on warming. This phase behaviour is particularly sensitive to interactions that perturb bilayer properties. In this study, ²H NMR was used to study the perturbation of bicellar lipid mixtures by two peptides (SP-B₆₃₋₇₈, a lung surfactant protein fragment and Magainin 2, an antimicrobial peptide) which are structurally similar. Particular attention was paid to the relation between peptide-induced perturbation and lipid composition. In bicellar dispersions containing only zwitterionic lipids (DMPC-d₅₄/DMPC/DHPC (3:1:1)) both peptides had little to no effect on the temperature at which coalescence to larger structures occurred. Conversely, in mixtures containing anionic lipids (DMPC-d₅₄/DMPG/DHPC (3:1:1)), both peptides modified bicellar phase behaviour. In mixtures containing SP-B₆₃₋₇₈, the presence of peptide decreased the temperature of the ribbon-like to extended lamellar phase transition. The addition of Magainin 2 to DMPCd₅₄/ DMPG/DHPC (3:1:1) mixtures, in contrast, increased the temperature of this transition and yielded a series of spectra resembling DMPC/DHPC (4:1) mixtures. Additional studies of lipid dispersions containing deuterated anionic lipids were done to determine whether the observed perturbation involved a peptide-induced separation of zwitterionic and anionic lipids. Comparison of DMPC/DMPG-d₅₄/DHPC (3:1:1) and DMPC-d₅₄/DMPG/DHPC (3:1:1) mixtures showed that DMPC and DMPG occupy similar environments in the presence of SP-B₆₃₋₇₈, but different lipid environments in the presence of Magainin 2. This might reflect the promotion of anionic lipid clustering by Magainin 2. These results demonstrate the variability of mechanisms of peptide-induced perturbation and suggest that lipid composition is an important factor in the peptide-induced perturbation of lipid structures.