The interaction of melittin with multicomponent lipid mixtures composed of phosphatidylcholine, sphingomyelin and phosphatidylserine or phosphatidylglycerol was investigated by measuring the intrinsic fluorescence of the peptide, steady state fluorescence anisotropy of, and Trp-fluorescence energy transfer to fluorescent analogs of the same phospholipids bearing the anthrylvinyl fluorophore in one of the aliphatic chains at various distances from the polar head group. Based on the finding that at high lipid/peptide ratio the peptide induces unequal changes in the fluorescence parameters of phospholipid probes differing structurally only in their polar head groups, it is concluded that melittin induces lipid demixing in its nearest environment. Comparison of the fluorescence energy transfer from Trp to different lipid probes indicates that the depth of penetration of melittin into the bilayer depends on the polar head group composition of the phospholipid matrix and that certain segments of the melittin chain display a specific affinity for a given lipid head group.

Animals↗

PubMedDate not supplied

Effect of the conformation of a peptide from gp41 on binding and domain formation in model membranes.

Binding of the peptide fragment 828-848 (P828), amino acid sequence RVIEVVQGACRAIRHIPRRIR, from the carboxy-terminal region of the envelope glycoprotein gp41 of human immunodeficiency virus type 1 (HIV-1) to membranes composed of a mixture of neutral and negatively charged phospholipids results in domain or cluster formation of the charged lipid. The conformation and dynamics of the peptide are investigated in solution and in the presence of sodium dodecyl sulphate (SDS) micelles using high resolution nuclear magnetic resonance (NMR) spectroscopy and circular dichroism (CD) spectropolarimetry. The CD results demonstrate that addition of either SDS, negatively charged phospholipid liposomes, or trifluoroethanol (TFE) induces a conformational transition of the peptide from a random coil or an extended chain in water to a more ordered structure with an estimated helical content of up to 60%. The structure of the peptide in a membrane mimetic SDS solution was investigated in detail using two-dimensional NMR. The measurements demonstrate the existence of a helical component in the peptide conformation in the SDS-bound state. The peptide most likely exists as an ensemble of conformations with exchange times between them which are fast on the chemical shift NMR time scale (10(-3) s). Simple neutralization of the six arginine sidechain charges does not cause the peptide to adopt an ordered structure. Thus, there is an additional requirement for the structural transition such as that resulting from constraint of the peptide on a surface, or localization of the peptide at the lipid-water interface where the polarity is lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Role of interactions at the lipid-water interface for domain formation.

The lipid-water interface is critical for the packing of lipid molecules in membranes. We have demonstrated that lateral phase separation in membranes can be driven by electrostatic interactions such as those involving charged lipid species and oppositely charged peptides, in addition to hydration effects at the lipid-water interface. By using nuclear magnetic resonance (NMR), circular dichroism and fluorescence spectroscopy we have shown that binding of a 21-amino acid peptide containing six positively charged arginine residues to mixed phosphatidylcholine (PC)/phosphatidylglycerol (PG) membranes results in a conformational change in the peptide from a random coil to a helical structure and causes the formation of domains of negatively charged PG. Binding of the peptide to PG membranes disorders the lipid hydrocarbon chains. The strength of lipid-peptide binding at the interface, the conformational change in the peptide, and domain formation with the negatively charged lipid are coupled energetically. The lipid-peptide association constant is lower for membranes containing 20 mol% PG in PC/PG mixtures than for 100% PG membranes. We suggest that one of the factors that lower the association constant in PC/PG membranes is entropic energy of formation of PG domains. Besides electrostatic interactions, hydration of lipids is important for domain formation. We have shown that dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine separate under conditions of decreased water activity. Furthermore, water activity controls lipid packing stress in the hydrocarbon core and the headgroups of membranes as demonstrated by induction of an inverse-hexagonal-to-lamellar phase transition in dioleoylphosphatidylethanolamine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMedDate not supplied

Dynamic lipid heterogeneity and receptor events.

Receptor occupation by specific ligands induces changes in the dynamic domain organization of surrounding lipids. Such changes were observed by measuring changes in the fluorescence parameters of fluorescent-labelled lipids incorporated into plasma membranes of intact cells, membrane vesicles or lipoprotein particles in response to specific binding of a broad range of biologically active agents, including drugs, prostaglandins, neuropeptides, antibodies and viruses. The high sensitivity of the fluorescence response allowed us to register changes in lipid heterogeneity induced in a multitude of discrete targets by transient weak binding of a single rapidly translocating molecule. To explain these observations a non-equilibrium model of ligand-receptor interaction based on low relaxation phenomena in heterogeneous lipid matrixes is proposed.

Humans↗

PubMedDate not supplied

Transient domains induced by influenza haemagglutinin during membrane fusion.

During low pH-induced fusion of influenza virus with erythrocytes we have observed differential dispersion of viral lipid and haemagglutinin (HA) into the erythrocyte membrane, and viral RNA into the erythrocyte using fluorescence video microscopy. The movement of both viral lipid and HA from virus to cell was restricted during the initial stages of fusion relative to free diffusion. This indicates the existence of relatively long-lived barriers to diffusion subsequent to fusion pore formation. Fluorescence anisotropy of phospholipid analogues incorporated into the viral membrane decreased when the pH was lowered to levels required for optimum fusion. This indicates that the restricted motion of viral membrane components was not due to rigidification of membrane lipids. The movement of HA from the fusion site was also assessed by photosensitized labelling by means of a fluorescent substrate (NBD-taurine) passing through the band 3 sialoglycoprotein (the erythrocyte anion transporter). We also examined the flow of lipid and aqueous markers during fusion of HA-expressing cells with labelled erythrocytes. During this cell-cell fusion, movement of lipid between fusing membranes begins before the fusion pore is wide enough to allow diffusion of aqueous molecules (M(r) > 500). The data indicate that HA is capable of creating domains in the membrane and controlling continuity of aqueous compartments which are bounded by such domains.

Cell Fusion↗