A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol % fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydroergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol % dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol% sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physical properties of the fluorescent sterol probe dehydroergosterol

Spectroscopic studies were performed on the fluorescent sterol probes ergosta-5,7,9(11),22-tetraen-3 beta-ol (dehydroergosterol) and cholesta-5,7,9(11)-trien-3 beta-ol (cholestatrienol). In most isotropic solvents, these molecules exhibited a single lifetime near 300 ps. Fluorescence lifetimes in 2-propanol were independent of emission wavelength and independent of excitation wavelength. Excited state behavior of these probes appears relatively simple. In isotropic solvents, dehydroergosterol fluorescence emission underwent at most a small Stokes shift as solvent polarity was modified. Time-resolved anisotropy decays indicated that dehydroergosterol decay was monoexponential, with rotational correlation times dependent on solvent viscosity. When incorporated into L-alpha-dimyristoylphosphatidylcholine liposomes at a concentration of 0.9 mol%, dehydroergosterol fluorescence lifetime decreased at the phase transition of this phospholipid indicating that the sterol probe was detecting physical changes of the bulk phospholipids. Furthermore, total fluorescence decays and anisotropy decays were sensitive to the environment of the sterol. Dehydroergosterol and cholestatrienol are thus useful probes for monitoring sterol behavior in biological systems.     

Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was investigated as a cholesterol analogue to examine sterol domains in and spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Fluorescence lifetime, acrylamide quenching analyses, and intermembrane exchange kinetics were consistent with the presence of at least two sterol domains in POPC. Fluorescence lifetime was determined by phase and modulation fluorescence spectroscopy and analyzed by nonlinear least-squares as well as continuous distributional analyses. Both methods demonstrated that pure dehydroergosterol in POPC SUV had two lifetime components (C) and fractional intensities (F) near C1 = 0.851 ns (F1 0.96) and C2 = 2.668 ns (F2 0.004). In contrast to component C1, the center of lifetime distribution, fractional intensity, and peak width of dehydroergosterol lifetime component C2 was dependent on the polarity of the medium and vesicle curvature. The sterol domain corresponding to dehydroergosterol component C2 was preferentially quenched by acrylamide. Acrylamide quenching of dehydroergosterol fluorescence demonstrated that the two lifetime components of dehydroergosterol were not due to transbilayer sterol domains with different lifetimes. In a spontaneous exchange assay not requiring separation of donor and acceptor SUV, the lifetime component C2, but not C1, shifted to a shorter lifetime with altered distributional width. The kinetics of these lifetime and distributional width changes best fitted a two-exponential function, with a fast exchange rate constant K1 = 0.0325 min-1, t1/2 = 21.3 min, and a slow rate constant k2 = 0.00275 min-1, t1/2 = 261 min. The fast exchanging pool correlates with the longer lifetime component C2. These kinetics were confirmed both by dehydroergosterol exchange measured with fluorescence intensity and by [3H]cholesterol exchange. In summary, lifetime, distributional width, acrylamide quenching, and classical exchange assay data are consistent with the presence of at least two pools of sterol in POPC SUV.     

A fluorescence and radiolabel study of sterol exchange between membranes

The fluorescent sterols delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) and delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) as well as [1,2-3H]cholesterol were utilized as cholesterol analogues to examine spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Exchange of fluorescent sterols was monitored at 24 degrees C by release from self-quenching of polarization from the time of mixing without separation of donor and acceptor vesicles. The polarization curve for 35 mol% sterol in POPC best fitted a two-exponential function, with a fast-exchange rate constant k1 = 0.0217 min-1, 1t1/2 = 32 min, size pool 1 = 12%, and a slow rate constant k2 = 2.91.10(-3) min-1, 2t1/2 = 238 min, size pool 2 = 88%. In addition to the above two exchangeable pools of sterol, the data were consistent with the presence of a slowly or nonexchangeable pool, 42% of total sterol, that was highly dependent on sterol content. These results were confirmed by simultaneous monitoring of [1,2-3H]cholesterol radioactivity and dehydroergosterol fluorescence intensity after separation of donor and acceptor vesicles by ion-exchange column chromatography. Thus, dehydroergosterol or cholestatrienol exchange as measured by fluorescence parameters (polarization and/or intensity) provides two new methods to follow cholesterol spontaneous exchange. These methods allow resolution and quantitation of a shorter exchange t1/2 near 30 min previously not reported. Thus, the cholesterol desorption rate from membranes may be faster than previously believed. In addition, the presence of a slowly non-exchangeable pool was confirmed.     

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence lifetime distributions of diphenylhexatriene-labeled phosphatidylcholine as a tool for the study of phospholipid-cholesterol interactions.

Fluorescence lifetimes of 1-palmitoyl-2-diphenylhexatrienylpro-pionyl-phosphatidylc hol ine in vesicles of palmitoyloleoyl phosphatidylcholine (POPC) (1:300, mol/mol) in the liquid crystalline state were determined by multifrequency phase fluorometry. On the basis of statistic criteria (chi 2red) the measured phase angles and demodulation factors were equally well fitted to unimodal Lorentzian, Gaussian, or uniform lifetime distributions. No improvement in chi 2red could be observed if the experimental data were fitted to bimodal Lorentzian distributions or a double exponential decay. The unimodal Lorentzian lifetime distribution was characterized by a lifetime center of 6.87 ns and a full width at half maximum of 0.57 ns. Increasing amounts of cholesterol in the phospholipid vesicles (0-50 mol% relative to POPC) led to a slight increase of the lifetime center (7.58 ns at 50 mol% sterol) and reduced significantly the distributional width (0.14 ns at 50 mol% sterol). Lifetime distributions of POPC-cholesterol mixtures containing greater than 20 mol% sterol were within the resolution limit and could not be distinguished from monoexponential decays on the basis of chi 2red. Cholesterol stabilizes and rigidifies phospholipid bilayers in the fluid state. Considering its effect on lifetime distributions of fluorescent phospholipids it may also act as a membrane homogenizer.     

Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranes and small unilamellar vesicles

This study examined the kinetics of sterol desorption from monolayer and small unilamellar vesicle membranes to 2-hydroxypropyl-beta-cyclodextrin. The sterols used include cholesterol, dehydroergosterol (ergosta-5,7,9,(11),22-tetraen-3beta-ol) and cholestatrienol (cholesta-5,7,9,(11)-trien-3beta-ol). Desorption rates of dehydroergosterol and cholestatrienol from pure sterol monolayers were faster (3.3-4.6-fold) than the rate measured for cholesterol. In mixed monolayers (sterol: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 30:70 mol%), both dehydroergosterol and cholestatrienol desorbed faster than cholesterol. clearly indicating a difference in interfacial behavior of these sterols. In vesicle membranes desorption of dehydroergosterol was slower than desorption of cholestatrienol, and both rates were markedly affected by the phospholipid composition. Desorption of sterols was slower from sphingomyelin as compared to phosphatidylcholine vesicles. Desorption of fluorescent sterols was also faster from vesicles prepared by ethanol-injection as compared to extruded vesicles. The results of this study suggest that dehydroergosterol and cholestatrienol differ from cholesterol in their membrane behavior, therefore care should be exercised when experimental data derived with these probes are interpreted.     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

A fluorescent sterol probe study of human serum low-density lipoproteins

The fluorescent sterol probe, ergosta-5,7,9,(11),22-tetraen-3 beta-ol (dehydroergosterol), was utilized as a cholesterol analog to label human serum low-density lipoproteins (LDL). Quenching of dehydroergosterol fluorescence by KI indicated that most of the fluorophore was either buried within the outer phospholipid monolayer of LDL or within the neutral lipid core of LDL. The steady-state anisotropy of dehydroergosterol in LDL detected the cholesteric core phase transition near 30 degrees C. Fluorescence lifetime decays for dehydroergosterol contained two components, both below and above the cholesteric phase transition, with the major lifetime component near 1 ns. Neither lifetime component underwent a detectable change in duration at the core phase transition temperature. Time-correlated fluorescence anisotropy decays of dehydroergosterol indicated a single rotational correlation time near 1.7 ns, which was unaffected by the core phase transition. Time-correlated anisotropy decays also suggested hindered rotation of dehydroergosterol in LDL. These results indicate that unesterified cholesterol is primarily located in the outer phospholipid monolayer of LDL, with the majority of cholesterol not in direct contact with the aqueous phase.     

Interaction of fluorescent delta 5,7,9(11),22-ergostatetraen-3 beta-ol with sterol carrier protein-2

The fluorescent sterol delta 5,7,9(11)-dehydroergostatetraen-3 beta-ol (dehydroergosterol) was used as an analogue of cholesterol to examine the molecular interaction of purified rat liver sterol carrier protein-2 (SCP-2) with sterol. The binding of dehydroergosterol to SCP-2 was evidenced by light scatter and by fluorescence polarization, lifetime, limiting anisotropy, and rotational relaxation time of dehydroergosterol. In addition, energy transfer efficiency from SCP-2 tryptophan to dehydroergosterol was 96%, indicating that the apparent distance, R, between the SCP-2 tryptophan (energy donor) and the dehydroergosterol (energy acceptor) was 13.7 A. Scatchard binding analysis of light scatter, lifetime, and energy transfer data all indicated a 1:1 molar stoichiometry with Kd = 1.2, 1.6, and 1.3 microM, respectively. SCP-2 enhanced the activity of microsomal acyl-CoA:cholesterol acyltransferase through transfer of [3H]cholesterol from donor palmitoyloleoyl phosphatidylcholine/cholesterol small unilamellar vesicles to rat liver microsomes containing the enzyme. A recently developed fluorescence assay utilizing dehydroergosterol fluorescence polarization (Nemecz, G., Fontaine, R. N., and Schroeder, F. (1988) Biochim. Biophys. Acta 948, 511-521; Nemecz, G., and Schroeder, F. (1988) Biochemistry 27, 7740-7749) was applied to examine the effect of SCP-2 on sterol exchange. In the absence of SCP-2, two spontaneously exchangeable sterol domains were observed in palmitoyloleoyl phosphatidylcholine/sterol (65:35 molar ratio) small unilamellar vesicles. SCP-2 enhanced the rate of exchange of the faster exchanging domain 2-fold. The transfer rate of the more slowly exchangeable sterol domain and the fraction of cholesterol represented by each domain were not affected. These results demonstrate the utility of dehydroergosterol to probe SCP-2 interactions with sterols and are indicative of a physiological role for SCP-2 as a soluble sterol carrier.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.     

A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.     

An InCytes from MBC Selection: Sterols Are Mainly in the Cytoplasmic Leaflet of the Plasma Membrane and the Endocytic Recycling Compartment in CHO Cells

The transbilayer distribution of many lipids in the plasma membrane and in endocytic compartments is asymmetric, and this has important consequences for signaling and membrane physical properties. The transbilayer distribution of cholesterol in these membranes is not properly established. Using the fluorescent sterols, dehydroergosterol and cholestatrienol, and a variety of fluorescence quenchers, we studied the transbilayer distribution of sterols in the plasma membrane (PM) and the endocytic recycling compartment (ERC) of a CHO cell line. A membrane impermeant quencher, 2,4,6-trinitrobenzene sulfonic acid, or lipid-based quenchers that are restricted to the exofacial leaflet of the plasma membrane only reduce the fluorescence intensity of these sterols in the plasma membrane by 15–32%. When the same quenchers have access to both leaflets, they quench 70–80% of the sterol fluorescence. Sterol fluorescence in the ERC is also quenched efficiently in the permeabilized cells. In microinjection experiments, delivery of quenchers into the cytosol efficiently quenched the fluorescent sterols associated with the PM and with the ERC. Quantitative analysis indicates that 60–70% of the PM sterol is in the cytoplasmic leaflet. This means that cholesterol constitutes ∼40 mol% of cytoplasmic leaflet lipids, which may have important implications for intracellular cholesterol transport and membrane domain formation.     

Cholesterol and ergosterol superlattices in three-component liquid crystalline lipid bilayers as revealed by dehydroergosterol fluorescence.

We have examined the fractional sterol concentration dependence of dehydroergosterol (DHE) fluorescence in DHE/cholesterol/dimyristoyl-L-alpha-phosphatidylcholine (DMPC), DHE/ergosterol/DMPC and DHE/cholesterol/dipalmitoyl-L-alpha-phosphatidylcholine (DPPC) liquid-crystalline bilayers. Fluorescence intensity and lifetime exhibit local minima (dips) whenever the total sterol mole fraction, irrespective of the DHE content, is near the critical mole fractions predicted for sterols being regularly distributed in hexagonal superlattices. This result provides evidence that all three of these naturally occurring sterols (e.g., cholesterol, ergosterol, and DHE) can be regularly distributed in the membrane and that the bulky tetracyclic ring of the sterols is the cause of regular distribution. Moreover, at the critical sterol mole fractions, the steady-state anisotropy of DHE fluorescence and the calculated rotational relaxation times exhibit distinct peaks, suggesting that membrane free volume reaches a local minimum at critical sterol mole fractions. This, combined with the well-known sterol condensing effect on lipid acyl chains, provides a new understanding of how variations in membrane sterol content change membrane free volume. In addition to the fluorescence dips/peaks corresponding to hexagonal superlattices, we have observed intermediate fluorescence dips/peaks at concentrations predicted by the centered rectangular superlattice model. However, the 22.2 mol% dip for centered rectangular superlattices in DHE/ergosterol/DMPC mixtures becomes diminished after long incubation (4 weeks), whereas on the same time frame the 22.2 mol% dip in DHE/cholesterol/DMPC mixtures remains discernible, suggesting that although all three of these sterols can be regularly distributed, subtle differences in sterol structure cause changes in lateral sterol organization in the membrane.     

Sterol domains in phospholipid membranes: dehydroergosterol polarization measures molecular sterol transfer.

The domain structure of cholesterol in membranes and factors affecting it are not well understood. A method, based on kinetics of delta 5,7,9,(11),22-erogostatetraen-3 beta-ol (dehydroergosterol) fluorescence polarization change and not requiring separation of donor and acceptor membranes, was used to examine sterol domains in three-component cholesterol:dehydroergosterol:phospholipid small unilamellar vesicles (SUV). A new mathematical data treatment was developed to provide a direct correlation between molecular sterol exchange and steady-state dehydroergosterol fluorescence polarization measurements. The method identified multiple kinetic pools of sterol in SUV: a small but rapidly exchanging pool, a predominant slowly exchanging pool, and a very slowly exchangeable (nonexchangeable) pool. The relative sizes of the pools and half-times of exchange were highly dependent on the presence of acidic phospholipids and on cytosolic proteins involved in sterol transfer. Thus, the method provides a direct measure of molecular sterol transfer between membranes without separating donor and acceptor membranes.     

Effect of cholesterol content on the structural and dynamic membrane properties of DMPC/DSPC large unilamellar bilayers

In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3 mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5 °C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3 mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3 mol% of Cho, the proportion of ordered domains reaches a maximum.     

Structure and dynamic properties of dehydroergosterol, 13-113-113-1

Dehydroergosterol has been widely used as a fluorescent analog of cholesterol for the investigation of lipoprotein, model membrane, and biological membrane structure. Although its synthesis was reported over fifty years ago, the complete structure and assignment of the three double bonds in the rings has not heretofore been firmly established. Therefore, dehydroergosterol was synthesized and purified by reverse phase high performance liquid chromatography. The proposed structure (^{8, 7, 9(11), 22}-ergostatetraen-3-o1), including the location of the double bond at ⁹⁽¹¹⁾, was confirmed by mass spectroscopy,¹H-NMR, and¹³C-NMR. In addition, a convenient assay for determination of impurities in dehydroergosterol preparations utilizing absorbance peak ratios is described. The spectroscopic properties of dehydroergosterol are highly dependent on solvent dielectric constant. Dehydroergosterol was incorporated into sonicated unilamellar vesicles composed of dimyristoylphosphatidylcholine or distearoylphosphatidylcholine. Arrhenius plots of dehydroergosterol fluorescence polarization indicated that the sterol was sensitive to the phase transitions of these phospholipids near 23° and 54°C, respectively. Differential polarized phase fluorescence and lifetime analysis were used to determine the dynamic properties of dehydroergosterol in the vesicles. At 37°C the limiting anisotropy, order parameter, and rotational rate of dehydroergosterol in dimyristoylphosphatidylcholine were 0.162, 0.65, and 0.71 nsec, respectively. The limiting anisotropy and order parameter, but not the rotational rate, of dehydroergosterol were sensitive to the temperature and/or the physical state of the phospholipid.     

Fluorescence studies of dehydroergosterol in phosphatidylethanolamine/phosphatidylcholine bilayers

Our previous fluorescence study has provided indirect evidence that lipid headgroup components tend to adopt regular, superlattice-like lateral distribution in fluid phosphatidylethanolamine/phosphatidylcholine (PE/PC) bilayers (, Biophys. J. 73:1967-1976). Here we have further studied this intriguing phenomenon by making use of the fluorescence properties of a sterol probe, dehydroergosterol (DHE). Fluorescence emission spectra, fluorescence anisotropy (r), and time-resolved fluorescence intensity decays of DHE in 1-palmitoyl-2-oleoyl-PC (POPC)/1-palmitoyl-2-oleoyl-PE (POPE) mixtures were measured as a function of POPE mole fraction (X(PE)) at 23 degrees C. Deviations, including dips or kinks, in the ratio of fluorescence peak intensity at 375 nm/fluorescence peak intensity at 390 nm (I(375)/I(390)), fluorescence decay lifetime (tau), or rotational correlation time (rho) of DHE versus PE composition plots were found at X(PE) approximately 0.10, 0.25, 0.33, 0.65, 0.75, and 0.88. The critical values at X(PE) approximately 0.33 and 0.65 were consistently observed for all measured parameters. In addition, the locations, but not the depth, of the dips for X(PE) < 0.50 did not vary significantly over 10 days of annealing at 23 degrees C. The observed critical values of X(PE) coincide (within +/-0.03) with some of the critical mole fractions predicted by a headgroup superlattice model proposing that the PE and PC headgroups tend to be regularly distributed in the plane of the bilayer. These results agree favorably with those obtained in our previous fluorescence study using dipyrenylPC and Laurdan probes and thus support the proposition that 1) regular arrangement within a domain exists in fluid PE/PC bilayers, and 2) superlattice formation may play a significant role in controlling the lipid composition of cellular membranes (, Proc. Natl. Acad. Sci. USA. 95:4964-4969). The present data provide new information on the physical properties of such superlattice domains, i.e., the dielectric environment and rotational motion of membrane sterols appear to change abruptly as the lipid headgroups exhibit regular superlattice-like distributions in fluid bilayers.     

Fluorescence of delta 5,7,9(11),22-ergostatetraen-3 beta-ol in micelles, sterol carrier protein complexes, and plasma membranes

The fluorescent sterol analogue delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was synthesized and purified by reverse-phase high-performance liquid chromatography. Dehydroergosterol in aqueous solution had a critical micelle concentration of 25 nM and a maximum solubility of 1.3 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of dehydroergosterol with purified rat liver squalene and sterol carrier protein (SCP). SCP increased the maximal solubility of dehydroergosterol in aqueous buffer. The fluorescence emission spectrum of dehydroergosterol was blue shifted upon addition of SCP. The fluorescence lifetime of dehydroergosterol in aqueous buffer was 2.3 ns; addition of SCP resulted in the appearance of a second lifetime component near 12.4 ns. The SCP increased the fluorescence polarization of monomeric dehydroergosterol in aqueous buffer from 0.033 to 0.086. Scatchard analysis of the binding data indicated that dehydroergosterol interacted with purified rat liver SCP with an apparent KD = 0.88 microM and Bmax = 4.8 microM. At maximal binding, 1.0 mol of dehydroergosterol was specifically bound per mole of SCP. The close molecular interaction of dehydroergosterol with SCP was also demonstrated by energy-transfer experiments. The intermolecular distance between SCP and bound dehydroergosterol was evaluated by fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene series of double bonds in dehydroergosterol. The transfer efficiency was 36%, and R, the apparent distance between the tyrosine energy donor and the dehydroergosterol energy acceptor, was 19 A. The significance of these data obtained in vitro for dehydroergosterol interaction with SCP was also tested in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)