Fusion Peptides Promote Formation of Bilayer Cubic Phases in Lipid Dispersions. An X-Ray Diffraction Study

AbstractSmall angle x-ray diffraction revealed a strong influence of the N-terminal influenza hemagglutinin fusion peptide on the formation of nonlamellar lipid phases. Comparative measurements were made on a series of three peptides, a 20-residue wild-type X-31 influenza virus fusion peptide, GLFGAIAGFIENGWEGMIDG, and its two point-mutant, fusion-incompetent peptides G1E and G13L, in mixtures with hydrated phospholipids, either dipalmitoleoylphosphatidylethanolamine (DPoPE), or monomethylated dioleoyl phosphatidylethanolamine (DOPE-Me), at lipid/peptide molar ratios of 200:1 and 50:1. All three peptides suppressed the HII phase and shifted the Lα–HII transition to higher temperatures, simultaneously promoting formation of inverted bicontinuous cubic phases, QII, which becomes inserted between the Lα and HII phases on the temperature scale. Peptide-induced QII had strongly reduced lattice constants in comparison to the QII phases that form in pure lipids. QII formation was favored at the expense of both Lα and HII phases. The wild-type fusion peptide, WT-20, was distinguished from G1E and G13L by the markedly greater magnitude of its effect. WT-20 disordered the Lα phase and completely abolished the HII phase in DOPE-Me/WT-20 50:1 dispersions, converted the QII phase type from Im3m to Pn3m and reduced the unit cell size from ∼38 nm for the Im3m phase of DOPE-Me dispersions to ∼15 nm for the Pn3m phase in DOPE-Me/WT-20 peptide mixtures. The strong reduction of the cubic phase lattice parameter suggests that the fusion-promoting WT-20 peptide may function by favoring bilayer states of more negative Gaussian curvature and promoting fusion along pathways involving Pn3m phase-like fusion pore intermediates rather than pathways involving HII phase-like intermediates

Salt Tolerance of Archaeal Extremely Halophilic Lipid Membranes

The membranes of extremely halophilic Archaea are characterized by the abundance of a diacidic phospholipid, archaetidylglycerol methylphosphate (PGP-Me), which accounts for 50-80 mol% of the polar lipids, and by the absence of phospholipids with choline, ethanolamine, inositol, and serine head groups. These membranes are stable in concentrated 3-5 m NaCl solutions, whereas membranes of non-halophilic Archaea, which do not contain PGP-Me, are unstable and leaky under such conditions. By x-ray diffraction and vesicle permeability measurements, we demonstrate that PGP-Me contributes in an essential way to membrane stability in hypersaline environments. Large unilamellar vesicles (LUV) prepared from the polar lipids of extreme halophiles, Halobacterium halobium and Halobacterium salinarum, retain entrapped carboxyfluorescein and resist aggregation in the whole range 0-4 m NaCl, similarly to LUV prepared from purified PGP-Me. By contrast, LUV made of polar lipid extracts from moderately halophilic and non-halophilic Archaea (Methanococcus jannaschii, Methanosarcina mazei, Methanobrevibacter smithii) are leaky and aggregate at high salt concentrations. However, adding PGP-Me to M. mazei lipids results in gradual enhancement of LUV stability, correlating with the PGP-Me content. The LUV data are substantiated by the x-ray results, which show that H. halobium and M. mazei lipids have dissimilar phase behavior and form different structures at high NaCl concentrations. H. halobium lipids maintain an expanded lamellar structure with spacing of 8.5-9 nm, which is stable up to at least 100 degrees C in 2 m NaCl and up to approximately 60 degrees C in 4 m NaCl. However, M. mazei lipids form non-lamellar structures, represented by the Pn3m cubic phase and the inverted hexagonal H(II) phase. From these data, the forces preventing membrane aggregation in halophilic Archaea appear to be steric repulsion, because of the large head group of PGP-Me, or possibly out-of-plane bilayer undulations, rather than electrostatic repulsion attributed to the doubly charged PGP-Me head group

Time-resolved x-ray diffraction and calorimetric studies at low scan rates: I. Fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases

The phase transitions in fully hydrated dipalmitoylphosphatidylcholine (DPPC) and DPPC/water/ethanol phases have been studied by lowangle time-resolved x-ray diffraction under conditions similar to those employed in calorimetry (scan rates 0.05-0.5°C/min and uniform temperature throughout the samples). This approach provides more adequate characterization of the equilibrium transition pathways and allows for close correlations between structural and thermodynamic data. No coexistence of the rippled gel (P(β')) and liquid-crystalline (L(α)) phases was found in the main transition of DPPC; rather, a loss of correlation in the lamellar structure, observed as broadening of the lamellar reflections, takes place in a narrow temperature range of ∼100 mK at the transition midpoint. Formation of a long-living metastable phase, denoted by P(β')(mst), differing from the initial P(β') was observed in cooling direction by both x-ray diffraction and calorimetry. No direct conversion of P(β')(mst) into P(β') occurs for over 24 h but only by way of the phase sequence P(β')(mst) → L(β') → P(β'). According to differential scanning calorimetry (DSC), the enthalpy of the P(β')(mst)-L(α) transition is by ∼5% lower than that of the P(β')-L(α) transition. The effects of ethanol (Rowe, E. S. 1983. Biochemistry. 22:3299-3305; Simon, S. A., and T. J. McIntosh. 1984. Biochim. Biophys. Acta 773:169-172) on the mechanism and reversibility of the DPPC main transition were clearly visualized. At ethanol concentrations inducing formation of interdigitated gel phase, the main transition proceeds through a coexistence of the initial and final phases over a finite temperature range. During the subtransition in DPPC recorded at scan rate 0.3°C/min, a smooth monotonic increase of the lamellar spacing from its subgel (L(c)) to its gel (L(β')) phase value takes place. The width of the lamellar reflections remains unchanged during this transformation. This provides grounds to propose a “sequential” relaxation mechanism for the subgel-gel transition which is not accompanied by growth of domains of the final phase within the initial one

Cubic phases in membrane lipids

On the basis of data obtained by time-resolved X-ray diffraction, we consider in the present article the occurrence and formation pathways of inverted bicontinuous cubic phases, or bilayer cubic phases, Q (II)(B) , in diluted dispersions of lipids representing major biomembrane lipid classes [phosphatidylethanolamines (PEs), mixtures of PEs and phosphatidylcholines (PCs) with other lipids, glycolipids]. We show that Q (II)(B) formation proceeds much more easily upon cooling from the H(II) phase than upon heating or isothermal conversion from the L(α) phase, thus identifying an indirect but faster route for Q (II)(B) phase induction in lipids. The data collected consistently show that the ability to convert into cubic phase upon temperature cycling appears to be a general property of all lipids exhibiting an L(α) ↔ H(II) phase transition. Admixtures of charged phospholipids, both anionic and cationic, strongly facilitate Q (II)(B) formation in PEs. Their effect may be attributed to increased electrostatic repulsion between the lipid bilayers that reduces the unbinding energy and facilitates the dissipation of the L(α) phase required for its conversion into bilayer cubic phase