Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains

Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.     

Raft-like domain formation in large unilamellar vesicles probed by the fluorescent phospholipid analogue, C12NBD-PC

The liquid-ordered/disordered-phase domain co-existence in large unilamellar vesicle membranes consisting of phosphatidylcholine:sphingomyelin (2:1) with different amounts of cholesterol has been examined using a concentration-dependent self-quenching of a single reporter molecule, C12NBD-PC. A temperature-dependent decrease of fluorescence intensity was associated with the expected formation and increase of l_o-phase membrane fraction in the vesicles. The result is consistent with exclusion of the fluorescent probe from the liquid-ordered phase which partitions preferentially into the liquid-disordered phase membrane domains. This leads to an increase of the local concentration of fluorophore in the liquid-disordered phase and a decrease of the quantum yield. This effect was used to obtain a quantitative estimation of the fraction of the vesicle membrane occupied by the liquid-ordered phase, Φ_o, as a function of temperature and cholesterol content between 0 and 45 mol%. The value of Φ_o was related to the assumed partition coefficient k_p of probe between liquid-ordered/disordered phases. For large unilamellar vesicles containing 20 and 4 mol% cholesterol and probe, respectively, with k_p = 0 (probe completely excluded from liquid-ordered phase), Φ_o = 0.16 and with k_p = 0.2, Φ_o = 0.2. The results are relevant to the action of detergent in the fractionation of detergent-resistant membrane from living cells.     

Phase-separation and domain-formation in cholesterol-sphingomyelin mixture: pulse-EPR oxygen probing

Membranes made of Chol/ESM (cholesterol/egg sphingomyelin) mixtures were investigated using saturation-recovery electron paramagnetic resonance spin-labeling methods, in which bimolecular collisions of relaxation agents (oxygen or nickel ethylenediamine diacetic acid) with spin labels are measured. Liquid-disordered (l_d) and liquid-ordered (l_o) phases, and cholesterol bilayer domains (CBDs) were discriminated and characterized by profiles of the oxygen transport parameter (OTP). In the l_d phase, coexisting with the l_o phase, the OTP profile is bell-shaped and lies above that in the pure ESM membrane. Changes in the OTP profile across the l_o phase are complex. When the l_o phase coexists with the l_d phase, the OTP profile is similar to that across the pure ESM membrane but with a steeper bell shape. With an increase in cholesterol concentration (up to the cholesterol-solubility threshold), the profile becomes rectangular, with low OTP values from the membrane surface to the depth of C9, and high values in the membrane center. This approximately threefold increase in the OTP occurs at the depth at which the rigid ring structure of cholesterol is immersed. Further addition of cholesterol and the formation of the CBD does not affect the OTP profile across the l_o phase. OTP values in the CBD are significantly lower than in the l_o phase.     

A detailed analysis of partial molecular volumes in DPPC/cholesterol binary bilayers

We examined the volumetric behavior of the dipalmitoylphosphatidylcholine (DPPC)/cholesterol binary bilayer system with high accuracy and more cholesterol concentrations to reveal the detailed molecular states in the liquid-disordered (L_d) phase, the liquid-ordered (L_o) phase and the gel phase. We measured the average specific volume of the binary bilayer at several temperatures by the neutral flotation method and calculated the average volume per molecule to estimate the partial molecular volumes of DPPC and cholesterol in each phase. As a result, we found that the region with intermediate cholesterol concentrations showed a more complicated behavior than expected from simple coexistence of L_d and L_o domains. We also measured fluorescence decay of trans-parinaric acid (tPA) added into the binary bilayer with more cholesterol concentrations to get further insight into the cholesterol-induced formation of the L_o phase. On the basis of these results we discuss the molecular interaction between DPPC and cholesterol molecule in the L_o phase and the manner of L_d/L_o phase coexistence.     

Glycosphingolipids in microdomain formation and their spatial organization

Plasma membranes regulate the influx and efflux of molecules across themselves and are also responsible for primary signal transduction between cells or within the same cell. Presence of lateral heterogeneity and the ability of reorganization are essential requirements for effective functioning of biomembranes. Lipid rafts are small, heterogeneous, dynamic domains enriched in glycosphingolipids, sphingomyelin and cholesterol, and profoundly influence membrane organization. Glycosphingolipids are inclined towards formation of liquid-ordered phases in membranes, both with and without cholesterol; they are therefore prime players in domain formation. Here, we discuss the role of glycosphingolipids in microdomain formation and their spatial organization within these rafts.     

Membrane microdomains: Role of ceramides in the maintenance of their structure and functions

Free-standing giant unilamellar vesicles were used to visualize the complex lateral heterogeneity, induced by ceramide in the membrane bilayer at micron scale using C₁₂-NBD-PC probe partitioning under the fluorescence microscope. Ceramide gel domains exist as leaf-like structures in glycerophospholipid/ceramide mixtures. Cholesterol readily increases ceramide miscibility with glycerophospholipids but cholesterol-ceramide interactions are not involved in the organization of the liquid-ordered phase as exemplified by sphingomyelin/cholesterol mixtures. Sphingomyelin stabilizes the gel phase and thus decreases ceramide miscibility in the presence of cholesterol. Gel/liquid-ordered/liquid-disordered phase coexistence was visualized in quaternary phosphatidylcholine/sphingomyelin/ceramide/cholesterol mixtures as occurrence of dark leaf-like and circular domains within a bright liquid phase. Sphingomyelin initiates specific ceramide-sphingomyelin interactions to form a highly ordered gel phase appearing at temperatures higher than pure ceramide gel phase in phosphatidylcholine/ceramide mixtures. Less sphingomyelin is engaged in formation of liquid-ordered phase leading to a shift in its formation to lower temperatures. Sphingomyelinase activity on substrate vesicles destroys micron L_o domains but induces the formation of a gel-like phase. The activation of phospholipase A₂ by ceramide on heterogeneous membranes was visualized. Changes in the phase state of the membrane bilayer initiates such morphological processes as membrane fragmentation, budding in and budding out was demonstrated.     

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 °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.     

Cholesterol-induced protein sorting: an analysis of energetic feasibility

The mechanism(s) underlying the sorting of integral membrane proteins between the Golgi complex and the plasma membrane remain uncertain because no specific Golgi retention signal has been found. Moreover one can alter a protein's eventual localization simply by altering the length of its transmembrane domain (TMD). M. S. Bretscher and S. Munro (SCIENCE: 261:1280-1281, 1993) therefore proposed a physical sorting mechanism based on the hydrophobic match between the proteins' TMD and the bilayer thickness, in which cholesterol would regulate protein sorting by increasing the lipid bilayer thickness. In this model, Golgi proteins with short TMDs would be excluded from cholesterol-enriched domains (lipid rafts) that are incorporated into transport vesicles destined for the plasma membrane. Although attractive, this model remains unproven. We therefore evaluated the energetic feasibility of a cholesterol-dependent sorting process using the theory of elastic liquid crystal deformations. We show that the distribution of proteins between cholesterol-enriched and cholesterol-poor bilayer domains can be regulated by cholesterol-induced changes in the bilayer physical properties. Changes in bilayer thickness per se, however, have only a modest effect on sorting; the major effect arises because cholesterol changes also the bilayer material properties, which augments the energetic penalty for incorporating short TMDs into cholesterol-enriched domains. We conclude that cholesterol-induced changes in the bilayer physical properties allow for effective and accurate sorting which will be important generally for protein partitioning between different membrane domains.     

Comparative calorimetric and spectroscopic studies of the effects of lanosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes

We carried out comparative DSC and Fourier transform infrared spectroscopic studies of the effects of cholesterol and lanosterol on the thermotropic phase behavior and organization of DPPC bilayers. Lanosterol is the biosynthetic precursor of cholesterol and differs in having three rather than two axial methyl groups projecting from the β-face of the planar steroid ring system and one axial methyl group projecting from the α-face, whereas cholesterol has none. Our DSC studies indicate that the incorporation of lanosterol is more effective than cholesterol is in reducing the enthalpy of the pretransition. Lanosterol is also initially more effective than cholesterol in reducing the enthalpies of both the sharp and broad components of the main phase transition. However, at sterol concentrations of 50 mol %, lanosterol does not abolish the cooperative hydrocarbon chain-melting phase transition as does cholesterol. Moreover, at higher lanosterol concentrations (~30–50 mol %), both sharp and broad low-temperature endotherms appear in the DSC heating scans, suggestive of the formation of lanosterol crystallites, and of the lateral phase separation of lanosterol-enriched phospholipid domains, respectively, at low temperatures, whereas such behavior is not observed with cholesterol at comparable concentrations. Our Fourier transform infrared spectroscopic studies demonstrate that lanosterol incorporation produces a less tightly packed bilayer than does cholesterol, which is characterized by increased hydration in the glycerol backbone region of the DPPC bilayer. These and other results indicate that lanosterol is less miscible in DPPC bilayers than is cholesterol, but perturbs their organization to a greater extent, probably due primarily to the rougher faces and larger cross-sectional area of the lanosterol molecule and perhaps secondarily to its decreased ability to form hydrogen bonds with adjacent DPPC molecules. Nevertheless, lanosterol does appear to produce a lamellar liquid-ordered phase in DPPC bilayers, although this phase is not as tightly packed as comparable cholesterol/DPPC mixtures.     

Bilayer interactions of pHLIP, a peptide that can deliver drugs and target tumors

The pH-dependent insertion of pHLIP across membranes is proving to be a useful property for targeting acidic tissues or tumors and delivering drugs attached to its C-terminus. It also serves as a model peptide for studies of protein insertion into membranes, so further elucidation of the insertion mechanism of pHLIP and its features is desirable. We examine how the peptide perturbs a model phosphatidylcholine membrane and how it associates with the lipid bilayer using an array of fluorescence techniques, including fluorescence anisotropy measurements of TMA-DPH anchored in bilayers, quenching of pHLIP fluorescence by brominated lipids and acrylamide, and measurements of energy transfer between aromatic residues of pHLIP and TMA-DPH. When pHLIP is bound to the surface of bilayers near neutral pH, the membrane integrity is preserved whereas the elastic properties of bilayers are changed as reported by an increase of membrane viscosity. When it is inserted, there is little perturbation of the lipids. The results also suggest that pHLIP can bind to the membrane surface in a shallow or a deep mode depending on the phase state of the lipids. Using parallax analysis, the change of the penetration depth of pHLIP was estimated to be 0.4 Å from the bilayer center and 2.8 Å from the membrane surface after the liquid-to-gel phase transition.     

Differential ability of cholesterol-enriched and gel phase domains to resist benzyl alcohol-induced fluidization in multilamellar lipid vesicles

Benzyl alcohol (BA) has a well-known fluidizing effect on both artificial and cellular membranes. BA is also likely to modulate the activities of certain membrane proteins by decreasing the membrane order. This phenomenon is presumably related to the ability of BA to interrupt interactions between membrane proteins and the surrounding lipids by fluidizing the lipid bilayer. The components of biological membranes are laterally diversified into transient assemblies of varying content and order, and many proteins are suggested to be activated or inactivated by their localization in or out of membrane domains displaying different physical phases. We studied the ability of BA to fluidize artificial bilayer membranes representing liquid-disordered, cholesterol-enriched and gel phases. Multilamellar vesicles were studied by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and trans-parinaric acid, which display different phase partitioning. Domains of different degree of order and thermal stability showed varying abilities to resist fluidization by BA. In bilayers composed of mixtures of an unsaturated phosphatidylcholine, a saturated high melting temperature lipid (sphingomyelin or phosphatidylcholine) and cholesterol, BA fluidized and lowered the melting temperature of the ordered and gel phase domains. In general, cholesterol-enriched domains were more resistant to BA than pure gel phase domains. In contrast, bilayers containing high melting temperature gel phase domains containing a ceramide or a galactosylceramide proved to be the most effective in resisting fluidization. The results of our study suggest that the ability of BA to affect the fluidity and lateral organization of the membranes was dependent on the characteristic features of the membrane compositions studied and related to the intermolecular cohesion in the domains.     

Liquid domains in vesicles investigated by NMR and fluorescence microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.     

Bilayer polarity and its thermal dependency in the ?o and ?d phases of binary phosphatidylcholine/cholesterol mixtures

Diverse variations in membrane properties are observed in binary phosphatidylcholine/cholesterol mixtures. These mixtures are nonideal, displaying single or phase coexistence, depending on chemical composition and other thermodynamic parameters. When compared with pure phospholipid bilayers, there are changes in water permeability, bilayer thickness and thermomechanical properties, molecular packing and conformational freedom of phospholipid acyl chains, in internal dipolar potential and in lipid lateral diffusion. Based on the phase diagrams for DMPC/cholesterol and DPPC/cholesterol, we compare the equivalent polarity of pure bilayers with specific compositions of these mixtures, by using the Py empirical scale of polarity. Besides the contrast between pure and mixed lipid bilayers, we find that liquid-ordered (?_o) and liquid-disordered (?_d) phases display significantly different polarities. Moreover, in the ?_o phase, the polarities of bilayers and their thermal dependences vary with the chemical composition, showing noteworthy differences for cholesterol proportions at 35, 40, and 45 mol%. At 20 °C, for DMPC/cholesterol at 35 and 45 mol%, the equivalent dielectric constants are 21.8 and 23.8, respectively. Additionally, we illustrate potential implications of polarity in various membrane-based processes and reactions, proposing that for cholesterol containing bilayers, it may also go along with the occurrence of lateral heterogeneity in biological membranes.     

N-palmitoyl-sulfatide participates in lateral domain formation in complex lipid bilayers

Sulfatides (galactosylceramidesulfates) are negatively charged glycosphingolipids that are important constituents of brain myelin membranes. These membranes are also highly enriched in galactosylceramide and cholesterol. It has been implicated that sulfatides, together with other sphingolipids, take part in lateral domain formation in biological membranes. This study was conducted to characterize the lateral phase behavior of N-palmitoyl-sulfatide in mixed bilayer membranes. Going from simple lipid mixtures with sulfatide as the only sphingolipid in a fluid matrix of POPC, to more complex membranes including other sphingolipids, we have examined 1) ordered domain formation with sulfatide, 2) sterol enrichment in such domains and 3) stabilization of the domains against temperature by the addition of calcium. Using two distinct phase selective fluorescent probes, trans-parinaric acid and cholestatrienol, together with a quencher in the fluid phase, we were able to distinguish between ordered domains in general and ordered domains enriched in sterol. We found that N-palmitoyl-sulfatide formed ordered domains when present as the only sphingolipid in a fluid phospholipid bilayer, but these domains did not contain sterol and their stability was unaffected by calcium. However, at low, physiologically relevant concentrations, sulfatide partitioned favorably into domains enriched in other sphingolipids and cholesterol. These domains were stabilized against temperature in the presence of divalent cations. We conclude that sulfatides are likely to affect the lateral organization of biomembranes.     

Importance of the phosphocholine linkage on sphingomyelin molecular properties and interactions with cholesterol; a study with phosphate oxygen modified sphingomyelin-analogues

We have characterized the molecular properties and membrane behavior of synthetically modified sphingomyelin analogues, modified on the oxygen connecting the phosphocholine group to the ceramide backbone. The oxygen was replaced with an S-atom (S-PSM), an NH-group (NH-PSM) or a CH₂-group (CH₂-PSM). Diphenylhexatriene and Laurdan anisotropy experiments showed that an S-linkage increased and NH- and CH₂-linkages decreased the stability of PSM-analogue bilayer membranes as compared to PSM. When the polarity of the interface was probed using Laurdan, S-PSM appeared to have a lower polarity as compared to PSM whereas NH-PSM and CH₂-PSM had higher polarities of their respective interfaces. Fluorescence quenching-studies with cholestatrienol showed that all compounds formed SM/cholesterol-rich domains. The S-PSM/cholesterol and PSM/cholesterol domains displayed a similar thermostability, whereas NH-PSM/cholesterol and CH₂-PSM/cholesterol domains were less thermostable. DSC on vesicles containing the PSM-analogues showed a more complex melting behavior as compared to PSM, whereas equimolar mixtures of the PSM-analogues and PSM showed almost ideal mixing with PSM for NH- and S-PSM. Our data show that the properties of the bond linking the phosphocholine head group to the 1-hydroxyl on the ceramide molecule is important for the stability of SM/SM and SM/cholesterol interactions.     

Detergents induce raft-like domains budding and fission from giant unilamellar heterogeneous vesicles: a direct microscopy observation

The effect of detergents on giant unilamellar vesicles (GUVs) composed of phosphatidylcholine, sphingomyelin and cholesterol and containing liquid-ordered phase (l(o)) domains was investigated. Such domains have been used as models for the lipid rafts present in biological membranes. The studied detergents included lyso-phosphatidylcholine, the product of phospholipase A2 activity, as well as Triton X-100 and Brij 98, i.e. detergents used to isolate lipid rafts as DRMs. Local external injection of each of the three detergents at subsolubilizing amounts promoted exclusion of l(o) domains from the GUV as small vesicles. The budding and fission processes associated with this vesiculation were interpreted as due to two distinct effects of the detergent. In this framework, the budding is caused by the initial incorporation of the detergent in the outer membrane leaflet which increases the spontaneous curvature of the bilayer. The fission is related to the inverted-cone molecular shape of the detergent which stabilizes positively curved structures, e.g. pores involved in vesicle separation. On the other hand, we observed in GUVs neither domain formation nor domain coalescence to be induced by the addition of detergents. This supports the idea that isolation of DRM from biological membranes by detergent-induced extraction is not an artifact. It is also suggested that the physico-chemical mechanisms involved in l(o) domain budding and fission might play a role in rafts-dependant endocytosis in cells.     

The diversity of the liquid ordered (Lo) phase of phosphatidylcholine/cholesterol membranes: a variable temperature multinuclear solid-state NMR and x-ray diffraction study

To investigate the properties of a pure liquid ordered (Lo) phase in a model membrane system, a series of saturated phosphatidylcholines combined with cholesterol were examined by variable temperature multinuclear (1H, 2H, 13C, 31P) solid-state NMR spectroscopy and x-ray scattering. Compositions with cholesterol concentrations>or=40 mol %, well within the Lo phase region, are shown to exhibit changes in properties as a function of temperature and cholesterol content. The 2H-NMR data of both cholesterol and phospholipids were used to more accurately map the Lo phase boundary. It has been established that the gel-Lo phase coexistence extends to 60 mol % cholesterol and a modified phase diagram is presented. Combined 1H-, 2H-, 13C-NMR, and x-ray scattering data indicate that there are large changes within the Lo phase region, in particular, 1H-magic angle spinning NMR and wide-angle x-ray scattering were used to examine the in-plane intermolecular spacing, which approaches that of a fluid Lalpha phase at high temperature and high cholesterol concentrations. Although it is well known for cholesterol to broaden the gel-to-fluid transition temperature, we have observed, from the 13C magic angle spinning NMR data, that the glycerol region can still undergo a "melting", though this is broadened with increasing cholesterol content and changes with phospholipid chain length. Also from 2H-NMR order parameter data it was observed that the effect of temperature on chain length became smaller with increasing cholesterol content. Finally, from the cholesterol order parameter, it has been previously suggested that it is possible to determine the degree to which cholesterol associates with different phospholipids. However, we have found that by taking into account the relative temperature above the phase boundary this relationship may not be correct.     

Direct measurement of phase coexistence in DPPC/cholesterol vesicles using Raman spectroscopy

The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid-cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (l(d)), solid-ordered (s(o)) and liquid-ordered (l(o)) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at approximately 27 and approximately 22 mol% for l(d)-l(o) and s(o)-l(o) coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule.     

Exclusion of a transmembrane-type peptide from ordered-lipid domains (rafts) detected by fluorescence quenching: extension of quenching analysis to account for the effects of domain size and domain boundaries

Sphingolipid/cholesterol-rich rafts are membrane domains thought to exist in the liquid-ordered state. To understand the rules governing the association of proteins with rafts, the behavior of a model membrane-inserted hydrophobic polypeptide (LW peptide, acetyl-K(2)W(2)L(8)AL(8)W(2)K(2)-amide) was examined. The distribution of LW peptide between coexisting ordered and disordered lipid domains was probed by measuring the amount of LW Trp fluorescence quenched by a nitroxide-labeled phospholipid that concentrated in disordered lipid domains. Strong quenching of the Trp fluorescence (relative to quenching in model membranes lacking domains) showed that LW peptide was concentrated in quencher-rich disordered domains and was largely excluded from ordered domains. Exclusion of LW peptide from the ordered domains was observed both in the absence and in the presence of 25-33 mol % cholesterol, indicating that the peptide is relatively excluded both from gel-state domains (which form in the absence of cholesterol) and from liquid-ordered-state domains (which form at high cholesterol concentrations). Because exclusion was also observed when ordered domains contained sphingomyelin in place of DPPC, or ergosterol in place of cholesterol, it appeared that this behavior was not strongly dependent on lipid structure. In both the absence and the presence of 25 mol % cholesterol, exclusion was also not strongly dependent upon the fraction of the bilayer in the form of ordered domains. To evaluate LW peptide behavior in more detail, an analysis of the effects of domain size and edges upon quenching was formulated. This analysis showed that quenching can be affected both by domain size and by whether a fluorescent molecule localized at domain edges. Its application to the quenching of LW peptide indicated that the peptide did not preferentially reside at the boundaries between ordered and disordered domains.     

Phases and domains in sphingomyelin–cholesterol membranes: structure and properties using EPR spin-labeling methods

EPR spin-labeling methods were used to investigate the order and fluidity of alkyl chains, the hydrophobicity of the membrane interior, and the order and motion of cholesterol molecules in coexisting phases and domains, or in a single phase of fluid-phase cholesterol/egg-sphingomyelin (Chol/ESM) membranes with a Chol/ESM mixing ratio from 0 to 3. A complete set of profiles for these properties was obtained for the liquid-disordered (l _d) phase without cholesterol, for the liquid-ordered (l _o) phase for the entire region of cholesterol solubility in this phase (from 33 to 66 mol%), and for the l _o-phase domain that coexists with the cholesterol bilayer domain (CBD). Alkyl chains in the l _o phase are more ordered than in the l _d pure ESM membrane. However, fluidity in the membrane center is greater. Also, the profile of hydrophobicity changed from a bell to a rectangular shape. These differences are enhanced when the cholesterol content of the l _o phase is increased from 33 to 66 mol%, with clear brake-points between the C9 and C10 positions (approximately where the steroid-ring structure of cholesterol reaches into the membrane). The organization and motion of cholesterol molecules in the CBD are similar to those in the l _o-phase domain that coexists with the CBD.