Aleksandre Fedorov

Sphingomyelin/Phosphatidylcholine/Cholesterol Phase Diagram: Boundaries and Composition of Lipid Rafts

The ternary system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is used to model lipid rafts. The phase behavior of the three binary systems PSM/POPC, PSM/cholesterol, and POPC/cholesterol is first experimentally determined. Phase coexistence boundaries are then determined for ternary mixtures at room temperature (23 degrees C) and the ternary phase diagram at that temperature is obtained. From the diagram at 23 degrees C and the binary phase diagrams, a reasonable expectation is drawn for the ternary phase diagram at 37 degrees C. Several photophysical methodologies are employed that do not involve detergent extraction, in addition to literature data (e.g., differential scanning calorimetry) and thermodynamic rules. For the ternary phase diagrams, some tie-lines are calculated, including the one that contains the PSM/POPC/ cholesterol 1:1:1 mixture, which is often used in model raft studies. The diagrams here described are used to rationalize literature results, some of them apparently discrepant, and to discuss lipid rafts within the framework of liquid-ordered/liquid-disordered phase coexistence.

Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study.

Large unilamellar vesicles of dimyristoylphosphatidylcholine/cholesterol mixtures were studied using fluorescence techniques (steady-state fluorescence intensity and anisotropy, fluorescence lifetime, and fluorescence resonance energy transfer (FRET)). Three compositions (cholesterol mole fraction 0.15, 0.20, and 0.25) and two temperatures (30 and 40 degrees C) inside the coexistence range of liquid-ordered (l(o)) and liquid-disordered (l(d)) phases were investigated. Two common membrane probes, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dimyristoylphosphatidylethanolamine (NBD-DMPE) and N-(lissamine(TM)-rhodamine B)-dimyristoylphosphatidylethanolamine (Rh-DMPE), which form a FRET pair, were used. The l(o)/l(d) partition coefficients of the probes were determined by individual photophysical measurements and global analysis of time-resolved FRET decays. Although the acceptor, Rh-DMPE, prefers the l(d) phase, the opposite is observed for the donor, NBD-DMPE. Accordingly, FRET efficiency decreases as a consequence of phase separation. Comparing the independent measurements of partition coefficient, it was possible to detect very small domains (<20 nm) of l(o) in the cholesterol-poor end of the phase coexistence range. In contrast, domains of l(d) in the cholesterol-rich end of the coexistence range have comparatively large size. These observations are probably related to different processes of phase separation, nucleation being preferred in formation of l(o) phase from initially pure l(d), and domain growth being faster in formation of l(d) phase from initially pure l(o).

Nonequilibrium phenomena in the phase separation of a two-component lipid bilayer.

Lipid bilayers composed of two phospholipids with significant acyl-chain mismatch behave as nonideal mixtures. Although many of these systems are well characterized from the equilibrium point of view, studies concerning their nonequilibrium dynamics are still rare. The kinetics of lipid demixing (phase separation) was studied in model membranes (large unilamellar vesicles of 1:1 dilauroylphosphatidylcholine (C(12) acyl chain) and distearoylphosphatidylcholine (C(18) acyl chain)). For this purpose, photophysical techniques (fluorescence intensity, anisotropy, and fluorescence resonance energy transfer) were applied using suitable probes (gel phase probe trans-parinaric acid and fluid phase probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-dilauroylphosphatidylethanolamine). The nonequilibrium situation was induced by a sudden thermal quench from a one-fluid phase equilibrium situation (higher temperature) to the gel/fluid coexistence range (lower temperature). We verified that the attainment of equilibrium is a very slow process (occurs in a time scale of hours), leading to large domains at infinite time. The nonequilibrium structure stabilization is due essentially to temporarily rigidified C(12) chains in the interface between gel/fluid domains, which decrease the interfacial tension by acting as surfactants. The relaxation process becomes faster with the increase of the temperature drop. In addition, heterogeneity is already present in the supposed homogeneous fluid mixture at the higher temperature.

Optoelectronic and structural properties of amorphous silicon–carbon alloys deposited by low-power electron-cyclotron resonance plasma-enhanced chemical-vapor deposition

The optoelectronic and structural properties of hydrogenated amorphous silicon-carbon alloys ~a-SiC:H! are studied over the entire compositional range of carbon content. The films are prepared using low-power electron-cyclotron resonance ~ECR! plasma-enhanced chemical vapor deposition. The carbon content was varied by using different methane ~or ethylene-!-to-silane gas phase ratios and by introducing the methane ~or ethylene! either remotely into the plasma stream or directly through the ECR source, together with the excitation gas ~hydrogen!. Regardless of the deposition conditions and source gases used, the optical, structural and transport properties of the a-SiC:H alloys followed simple universal dependencies related to changes in the density of states associated with their structural disorder. The deep defect density from photothermal deflection spectroscopy, the ESR spin density, the steady state and the transient photoluminescence, the dark and photoconductivity, the temperature of the hydrogen evolution peaks and the bonding from infrared spectroscopy are correlated to the Urbach tail energy, the B factor of the Tauc plot and E04 ~defined as the energy at which the absorption coefficient is equal to 10 4 cm 21 !. Silicon-rich and carbon-rich regions with very different properties, corresponding approximately to carbon fractions below and above 0.5, respectively, can be distinguished. The properties of the ECR a-SiC:H alloys are compared with those of alloys deposited by rf glow discharge.

Fluorescence quenching with exponential distance dependence: Application to the external heavy-atom effect

A model for fluorescence quenching with exponential distance dependence is developed and applied to external heavy-atom quenching. The systems studied are C70-bromobenzene and phenanthrene-iodide, in liquid solutions at room temperature and in rigid glasses at 77 K. The predicted parameter correlation is observed in the two systems, which correspond to two extreme and opposite situations, strong and weak quenching. A detailed analysis of the fitted parameters allows the determination of the effective Bohr radii L and of the intrinsic unimolecular rate constants for quenching at contact, k0. The unusually high value of L for the C70-bromobenzene pair is tentatively attributed to the extended size of the external part of the π orbitals of the fullerene. The room temperature quenching rate constant computed with the determined low-temperature parameters is shown to be in good agreement with the experimental one for both systems studied.

Cholesterol Modulates the Organization of the γM4 Transmembrane Domain of the Muscle Nicotinic Acetylcholine Receptor

A 28-mer gammaM4 peptide, obtained by solid-state synthesis and corresponding to the fourth transmembrane segment of the nicotinic acetylcholine receptor gamma-subunit, possesses a single tryptophan residue (Trp453), making it an excellent model for studying peptide-lipid interactions in membranes by fluorescence spectroscopy. The gammaM4 peptide was reconstituted with synthetic lipids (vesicles of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, i.e., POPC) rich and poor in cholesterol and analyzed using steady-state and time-resolved fluorescence techniques. The decrease in gammaM4 intrinsic fluorescence lifetime observed upon incorporation into a cholesterol-rich lo phase could be rationalized on the basis of a dynamic self-quenching owing to the formation of peptide-rich patches in the membrane. This agrees with the low Förster type resonance energy transfer efficiency from the Trp453 residue to the fluorescent cholesterol analog, dehydroergosterol, in the lo phase. In the absence of cholesterol the gammaM4 nicotinic acetylcholine receptor peptide is randomly distributed in the POPC bilayer with its hydrophobic moiety matching the membrane thickness, whereas in the presence of cholesterol the increase in the membrane thickness and variation of the material properties favor the formation of peptide-enriched patches, i.e., interhelix interaction energy is essential for obtaining a stabilized structure. Thus, the presence of a cholesterol-rich, ordered POPC phase drives the organization of peptide-enriched patches, in which the gammaM4 peptide occupies approximately 30% of the patch area.

Interaction of α-Melanocyte Stimulating Hormone with Binary Phospholipid Membranes: Structural Changes and Relevance of Phase Behavior

The interaction of alpha-melanocyte stimulating hormone (alpha-MSH) with negatively charged binary membrane systems composed of either 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)], (DMPC/DMPG) or DMPC/1,2-dimyristoyl-sn-glycero-3-phosphate (DMPC/DMPA), both at a 3:1 ratio, was studied using complementary techniques (differential scanning calorimetry, infrared and ultraviolet absorption spectroscopy, and steady-state and time-resolved fluorescence). The peptide structure in buffer, at medium to high concentrations, is a mixture of aggregated beta-strands and random coil, and upon increasing the temperature the random coil configuration becomes predominant. At low concentrations (micromolar) there are essentially no aggregates. When in interaction with the lipidic systems this transition is prevented and the peptide is stabilized in a specific conformation different from the one in solution. The incorporation of alpha-MSH into phosphatidic acid-containing systems produced a significant alteration of the calorimetric data. Lateral heterogeneity can be induced by the peptide in the DMPA-containing mixture, at variance with the one of DMPG. In addition, the lipid/water partition coefficient for the peptide in the presence of DMPC/DMPA is greater in the gel phase as compared to the fluid phase. From the high values of limiting anisotropies it can be concluded that the peptide presents a very reduced rotational dynamics when in interaction with the lipids, pointing out to a strong interaction. Overall, these results show that the structure and stability of alpha-MSH in a negatively charged membrane environment are substantially different from those of the peptide in solution, being stabilized in a specific conformation that could be important to eliciting its biological activity.

Synthesis and fluorescence properties of [60] and [70]fullerene–coumarin dyads: Efficient dipole–dipole resonance energy transfer from coumarin to fullerene

New [60] and [70]fullerene–coumarin dyads were prepared by covalently linking a coumarin dye to a fullerene (C60 or C70) via a 1,3-dipolar cycloaddition reaction of azomethine ylides or by a cyclopropanation reaction of fullerenes with malonate derivatives. These dyads were spectroscopically characterized and their fluorescence properties studied. Fluorescence quenching of the coumarin fluorophore in the dyads results from efficient dipole–dipole resonance energy transfer from the coumarin moiety to the fullerene moiety.

Picosecond time-resolved and steady-state studies of the polarization of the fluorescence of C60 and C70

From a measurement of the time-resolved and steady-state fluorescence anisotropies of C60 and C70 in low-temperature organic glasses, it is concluded that the fluorescence of C60 is intrinsically unpolarized, and that the emission transition moment of C70 is located in the molecular xy plane. Also for C70, but at higher energies, some transitions occur mainly along the z molecular axis.

Effect of halogenated compounds on the photophysics of C70 and a monoadduct of C70: Some implications on optical limiting behaviour

The fluorescence spectra, quantum yields and lifetimes of C70 and a pseudo-dihydro derivative (C70R) have been measured in a wide range of solvents at room temperature. This information is important for the development of reverse saturable absorbers. Phosphorescence spectra and phosphorescence lifetimes were also measured at low temperature. The fluorescence is subject to quenching by halogenated compounds. The eAciency of quenching follows the order I > Br Cl. The nature of the quenching is shown to vary, with chlorinated compounds exhibiting static quenching of fullerene fluorescence, owing to nonfluorescent complex formation, whilst those compounds containing bromine and iodine exhibit dynamic quenching due to the external heavy-atom eAect, that increases the intersystem crossing rate constant in the fluorophore‐perturber complex. This constant is evaluated by an original method from the bimolecular quenching rate constants. The phosphorescence quantum yield of both fullerenes at 77 K slightly increases in the presence of iodobenzene, in spite of a strong decrease in phosphorescence lifetime. The marked increase of the intersystem crossing rate constant in concentrated solutions owing to the external heavy-atom eAect is of interest for the application of fullerenes as fast-responding optical limiters (reverse saturable absorbers) of intense laser pulses, even in cases where the triplet quantum yield is of the order of unity. ” 2001 Elsevier Science B.V. All rights reserved.