Effect of ring-substituted oxysterols on the phase behavior of dipalmitoylphosphatidylcholine membranes

Oxysterols are oxygenated derivatives of cholesterol that form a class of potent regulatory molecules with diverse biological activity. Given the implications of oxysterols in several physiological/pathophysiological pathways of human diseases, it is important to identify how their presence affects the biophysical properties of cell membranes. In this article we first describe the structure, formation, and biological functions of oxysterols, and previous work on the effect of these molecules on the structure and phase behavior of lipid membranes. We then present results of our X-ray diffraction experiments on aligned multilayers of dipalmitoylphosphatidylcholine (DPPC) membranes containing ring-substituted oxysterols. The effect of these molecules on the phase behavior of DPPC membranes is found to be very similar to that of cholesterol. All the oxysterols studied induce a modulated phase in DPPC membranes, similar to that reported in DPPC–cholesterol membranes. However, some differences are observed in the ability of these molecules to suppress the main transition of the lipid and to induce chain ordering, which might be related to differences in their orientation in the bilayer.     

Membrane and protein interactions of oxysterols

PURPOSE OF REVIEW: Oxysterols, oxidation products of cholesterol, mediate numerous and diverse biological processes. The objective of this review is to explain some of the biochemical and cell biological properties of oxysterols based on their membrane biophysical properties and their interaction with integral and peripheral membrane proteins. RECENT FINDINGS: According to their biophysical properties, which can be distinct from those of cholesterol, oxysterols can promote or inhibit the formation of membrane microdomains or lipid rafts. Oxysterols that inhibit raft formation are cytotoxic. The stereo-specific binding of cholesterol to sterol-sensing domains in cholesterol homeostatic pathways is not duplicated by oxysterols, and some oxysterols are poor substrates for the pathways that detoxify cells of excess cholesterol. The cytotoxic roles of oxysterols are, at least partly, due to a direct physical effect on membranes involved in cholesterol-induced cell apoptosis and raft mediated cell signaling. Oxysterols regulate cellular functions by binding to oxysterol binding protein and oxysterol binding protein-related proteins. Oxysterol binding protein is a sterol-dependent scaffolding protein that regulates the extracellular signal-regulated kinase signaling pathway. According to a recently solved structure for a yeast oxysterol binding protein-related protein, Osh4, some members of this large family of proteins are likely sterol transporters. SUMMARY: Given the association of some oxysterols with atherosclerosis, it is important to identify the mechanisms by which their association with cell membranes and intracellular proteins controls membrane structure and properties and intracellular signaling and metabolism. Studies on oxysterol binding protein and oxysterol binding protein-related proteins should lead to new understandings about sterol-regulated signal transduction and membrane trafficking pathways in cells.     

The polar nature of 7-ketocholesterol determines its location within membrane domains and the kinetics of membrane microsolubilization by apolipoprotein A-I

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.     

Interaction of two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol,with phosphatidylcholine and sphingomyelin in model membranes

Oxidized analogs of cholesterol (oxysterols) are produced through both enzymatic and non-enzymatic pathways and have been shown to perturb membrane properties in vitro and in vivo. In the present study, the membrane behavior of two naturally occurring oxysterols, 25-hydroxycholesterol and 7-ketocholesterol, was examined in two model systems. The presence of an additional oxygen moiety was found to alter membrane properties compared to native cholesterol and to each other in lipid monolayers, composed of either pure sterol or sterol–glycerophospholipid and sterol–sphingomyelin binary films, as well as in mixed multilamellar vesicles. The ability of oxysterols to condense phosphatidylcholine and sphingomyelin films, their capacity to cause changes in in-plane elasticity moduli, and their propensity to form detergent-resistant membrane domains were all found to be dependant on the location of the oxygen functionality in the oxysterol, the chemical nature of the phospholipid in the model systems, and the oxysterol/phospholipid ratio in the membrane. The findings described in this study with respect to their biophysical/biophysiological implications provide additional insight into the activity of cytotoxic oxysterols in model membranes.     

Interaction of ceramides with phosphatidylcholine, sphingomyelin and sphingomyelin/cholesterol bilayers

Ceramides (Cers) may exert their biological activity through changes in membrane structure and organization. To understand this mechanism, the effect of Cer on the biophysical properties of phosphatidylcholine, sphingomyelin (SM) and SM/cholesterol bilayers was determined using fluorescence probe techniques. The Cers were bovine brain Cer and synthetic Cers that contained a single acyl chain species. The phospholipids were 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1,2-dipalmitoyl-sn-glyero-3-phosphocholine (DPPC) and bovine brain, egg yolk and bovine erythrocyte SM. The addition of Cer to POPC and DPPC bilayers that were in the liquid-crystalline phase resulted in a linear increase in acyl chain order and decrease in membrane polarity. The addition of Cer to DPPC and SM bilayers also resulted in a linear increase in the gel to liquid-crystalline phase transition temperature (T(M)). The magnitude of the change was dependent upon Cer lipid composition and was much higher in SM bilayers than DPPC bilayers. The addition of 33 mol% cholesterol essentially eliminated the thermal transition of SM and SM/Cer bilayers. However, there is still a linear increase in acyl chain order induced by the addition of Cer. The results are interpreted as the formation of DPPC/Cer and SM/Cer lipid complexes. SM/Cer lipid complexes have higher T(M)s than the corresponding SM because the addition of Cer reduces the repulsion between the bulky headgroup and allows closer packing of the acyl chains. The biophysical properties of a SM/Cer-rich bilayer are dependent upon the amount of cholesterol present. In a cholesterol-poor membrane, a sphingomyelinase could catalyze the isothermal conversion of a liquid-crystalline SM bilayer to a gel phase SM/Cer complex at physiological temperature.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

The Anti-inflammatory Drug Indomethacin Alters Nanoclustering in Synthetic and Cell Plasma Membranes

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase seperation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Phosphatidylcholine and sphingomyelin containing an elaidoyl fatty acid can form cholesterol-rich lateral domains in bilayer membranes

Elaidic acid is a trans-fatty acid found in many food products and implicated for having potentially health hazardous effects in humans. Elaidic acid is readily incorporated into membrane lipids in vivo and therefore affects processes regulating membrane physical properties. In this study the membrane properties of sphingomyelin and phosphatidylcholine containing elaidic acid (N-E-SM and PEPC) were determined in bilayer membranes with special emphasis on their interaction with cholesterol and participation in ordered domain formation. In agreement with previous studies the melting temperatures were found to be about 20 degrees C lower for the elaidoyl than for the corresponding saturated lipids. The trans-unsaturation increased the polarity at the membrane-water interface as reported by Laurdan fluorescence. Fluorescence quenching experiments using cholestatrienol as a probe showed that both N-E-SM and PEPC were incorporated in lateral membrane domains with sterol and saturated lipids. At low temperatures the elaidoyl lipids were even able to form sterol-rich domains without any saturated lipids present in the bilayer. We conclude from this study that the ability of N-E-SM and PEPC to form ordered domains together with cholesterol and saturated phospho- and sphingolipids in model membranes indicates that they might have an influence on raft formation in biological membranes.     

Phase separation is induced by phenothiazine derivatives in phospholipid/sphingomyelin/cholesterol mixtures containing low levels of cholesterol and sphingomyelin

Lipid rafts are membrane structures enriched in cholesterol, sphingomyelin and glycolipids. In majority raft-mimicking model systems high contents of cholesterol and sphingomyelin (approximately 30 mol%) are used. Existence of raft-like structures was, however, reported also in model and natural membranes containing low levels of cholesterol and sphingomyelin. In the present work differential scanning calorimetry and fluorescence spectroscopy with the use of Laurdan probe was employed to demonstrate the existence of phase separation in model systems containing DPPC with addition of 5 mol% or 10 mol% of both cholesterol and sphingomyelin. Additionally, the influence of three phenothiazine derivatives on phase separation in mixed DPPC/cholesterol/sphingomyelin bilayers was investigated. Chlorpromazine, thioridazine and trifluoperazine were able to induce phase separation in DPPC and DPPC/cholesterol/sphingomyelin bilayers in temperatures below lipid main phase transition. However, only trifluoperazine induced phase separation in temperatures close to or above main phase transition. Trifluoperazine also induced phase separation in bilayers composed of egg yolk PC or DOPC mixed with cholesterol and sphingomyelin. We concluded that presence of lipid domains can be observed in model membranes containing low levels of cholesterol and sphingomyelin. Among three phenothiazine derivatives studied, only trifluoperazine was able to induce a permanent phase separation in phosphatidylcholine/cholesterol/sphingomyelin systems.     

Effect of ganglioside-GM1 on the order of phosphatidylcholine-cholesterol multilamellar liposomes. A fluorescence polarization study

The effect(s) of bovine brain ganglioside-GM1 on the order of phosphatidylcholine-cholesterol membranes were studied using steady-state fluorescence polarization (FPZ) techniques with 1,6-diphenyl-1,3,5-hexatriene (DPH) as the membrane probe. In the absence of cholesterol, GM1 (30 mol%) increases both membrane order and the phase transition temperature of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) membranes. However, in the presence of cholesterol (0.3 or 0.5, cholesterol/phospholipid molar ratio), GM1 significantly decreases steady-state anisotropy (rs) at temperatures above the Tm for the particular phospholipid. This effect may, in part relate to a dilution of membrane cholesterol and is shared by bovine brain sphingomyelin (SM). GM1 (30 mol%) increases the order of 1-palmityl-2-oleyl-PC (POPC) membranes. However, in the presence of cholesterol (0.3 molar ratio) GM1 neither increases or decreases order. Thus, in cholesterol containing artificial membranes, the effect of GM1 depends on the phosphatidylcholine (PC) fatty acid composition and may not be evident from the effect of GM1 on pure PC membranes.     

Relationship between sterol/steroid structure and participation in ordered lipid domains (lipid rafts): implications for lipid raft structure and function

The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.     

Changes in membrane fluidity evoked by organophosphorus insecticide bromfenvinfos and its methylated analogue

The interaction of organophosphorus insecticides bromfenvinfos and methyl bromfenvinfos with model and native membranes was investigated by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), a probe located in the hydrophobic core of the bilayer and 1,3-bis-(1-pyrene)propane, a probe distributed in the outer region of the bilayer. DPH reported a broadening of the transition profile and solidifying effects in the fluid phase of liposomes formed from dimyristoyl (DMPC), dipalmitoyl (DPPC), and distearoyl (DSPC) phosphatidylcholine in the presence of the insecticides. A shift of the transition temperature towards a lower temperature was observed in DPPC- and DSPC-bromfenvinfos-treated vesicles. Py(3)Py detected an ordering effect of the insecticides in the fluid state of the lipids and abolished pre-transition in DPPC and DSPC vesicles. These results suggest that the insecticides localize in the co-operative region of the bilayer. Cholesterol added to DMPC decreased the influence of the insecticides as reported by both DPH and Py(3)Py. The effect of the insecticides on the fluidity of some native membranes, namely erythrocytes, lymphocytes, brain microsomes, and sarcoplasmic reticulum, depended on the cholesterol content in these membranes, the higher the cholesterol content, the smaller the solidifying effect. The physical mechanism of action of the insecticides on membrane lipids can be similar to that of cholesterol. All observed effects were more pronounced for bromfenvinfos than for its methylated analogue which correlates with the toxicity of these compounds for mammals.     

Oxysterols: biological activities and physicochemical studies.

To improve the understanding of the various biological activities of oxysterols (oxygenated derivatives of cholesterol), studies of their physicochemical properties have been undertaken. Oxysterols modify membrane dynamic properties which consequently trigger several biological effects. Despite the presence of at least one oxygenated group in addition to the C3 beta-hydroxyl, oxysterols insert perfectly into the lipidic bilayer of the membrane inducing a condensing effect similar to, but less potent than, that of cholesterol. In biological membranes oxysterols probably interact with membrane components as they are not easily exchanged after their incorporation into the cell membrane. These lipid-protein interactions are probably crucial for the expression of the biological activities of the oxysterols.     

Effect of chronic endothelin blockade on PKC isoform distribution in mesenteric arteries from diabetic rats

Rigid plaques containing protein particles in plasma membrane build on the apical surface of the mammalian urothelium. We have previously shown that dietary fats modified the fatty acid profile as well as the fluorescence anisotropy of rat urothelial plasma membranes. In this study, we have further examined the proportion of phosphatidylcholine, phosphatidylethanolamine, cerebrosides, sulfatides and cholesterol in detergent resistant (DRM) and soluble (DSM) plasma membrane fractions as well as the properties of the particles. Four groups of weaned rats were fed for 12 weeks on a commercial diet (control), or on a formula containing 5% (w/w) of corn oil, fish oil or olein. The control DRM behaved as a distinctive domain since it was enriched in cholesterol and glycosphingolipids. DSM showed higher levels of phosphatidylcholine and phosphatidylethanolamine with respect to DRM. On the other hand, the lipid distributions were affected by the diets. Homogeneous lipid distributions between DSM and DRM were found in olein membranes, suggesting a decreased potential formation of lipid domains. In addition, properties of the uroplakins were altered by dietary treatments. Thus, uroplakins (UP) Ia, Ib, II and III observed by SDS-PAGE, were in lower proportions (mainly olein) than in controls. Moreover, a higher proportion of UPIII was cross-linked to UPIII and UPIb in olein treatment than in control. Meanwhile, only cross-linking to UPIII or UPIb was altered in corn and fish diets, respectively. These results suggest a role of the lipids in the establishment of the uroplakin interactions. Thus, specific dietary fats may have important functional implications. (Mol Cell Biochem 271: 69–75, 2005)     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Fluorescence and thermotropic studies of the interactions of PEI-cholesterol based PEI-chol lipopolymers with dipalmitoyl phosphatidylcholine membranes

Numerous PEI derived polymers have been explored for their use in gene delivery. Nine PEI-chol lipopolymers based on cholesterol grafting on three polyethyleneimines (PEI) of different molecular weights have been synthesized. Firstly their aggregation behavior has been studied using transmission electron microscopy and then their interactions with l-α-dipalmitoyl phosphatidylcholine (DPPC) membranes have been examined using fluorescence anisotropy and differential scanning calorimetry (DSC). These PEI-chol lipopolymers are found to quench the chain motion of the acyl chains of DPPC, when incorporated in membranes, depending upon the cholesterol grafting on PEI. These PEI-chol lipopolymers act as filler molecules in membranes. Electron microscopy shows the different aggregation behavior of these new PEI-chol lipopolymers depending upon the molecular weight of PEI and percentage of cholesterol grafting. Detailed analysis of the fluorescence anisotropy and DSC data indicate that the nature of perturbation induced by PEI-chol lipopolymers is dependent upon the molecular weight of the PEI used and the % of cholesterol grafting on PEI. In general, PEI-chol lipopolymers rigidify the liquid-crystalline phase of the membranes without any noticeable effect on the gel phase unlike natural cholesterol, which is known to fluidize the gel phase of the membranes.     

Influence of cholesterol and ergosterol on membrane dynamics using different fluorescent reporter probes

Ergosterol is an evolutionary precursor of cholesterol and is the major sterol present in lower eukaryotes. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is still at a very early stage. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing fluorescent reporter probes pyrene and TMA-DPH. These results show, for the first time, the important differences on the effect of cholesterol and ergosterol in short-range ordering (reported by TMA-DPH) and long-range dynamics (reported by pyrene). In addition, pyrene vibronic peak intensity ratio provides information on polarity of the microenvironment experienced by the probe. These novel results are relevant in the context of membrane domains in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Fluorescence lifetimes of carbocyanine lipid analogues in phospholipid bilayers

The fluorescence lifetimes for the 1,1'-dialkyl-3,3,3',3'-tetramethylindocarbocyanine (CNdiI) dyes (N = 12, 18, and 22) in a variety of lipid bilayer membranes were measured. Effects of bilayer physical state, probe chain length, probe concentration, charge, lipid head group, and cholesterol concentration were examined. Even in single-phase membranes these probes did not exhibit single-exponential decays. Rather, the data were fit by biexponential decays with lifetimes of approximately 0.3-0.4 and approximately 0.9-1.3 ns with no significant improvement in chi 2 convergence with the addition of a third component. Average lifetimes were dependent upon lipid phase and to a lesser degree surface charge and the phospholipid head group. In dipalmitoyl-phosphatidylcholine (DPPC)-cholesterol membranes, the C18diI lifetime was sensitive to membrane reorganizations at both 20 and approximately 33 mol % cholesterol. In egg phosphatidylcholine (EPC) bilayers, the C18diI lifetime was essentially independent of its concentration below 1:10(3).