Phase Separation in Lipid Membranes

Cell membranes show complex behavior, in part because of the large number of different components that interact with each other in different ways. One aspect of this complex behavior is lateral organization of components on a range of spatial scales. We found that lipid-only mixtures can model the range of size scales, from approximately 2 nm up to microns. Furthermore, the size of compositional heterogeneities can be controlled entirely by lipid composition for mixtures such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol or sphingomyelin (SM)/DOPC/POPC/cholesterol. In one region of special interest, because of its connection to cell membrane rafts, nanometer-scale domains of liquid-disordered phase and liquid-ordered phase coexist over a wide range of compositions.     

Kinetics of cholesterol extraction from lipid membranes by methyl-β-cyclodextrin—A surface plasmon resonance approach

The kinetics of cholesterol extraction from cellular membranes is complex and not yet completely understood. In this paper we have developed an experimental approach to directly monitor the extraction of cholesterol from lipid membranes by using surface plasmon resonance and model lipid systems. Methyl-β-cyclodextrin was used to selectively remove cholesterol from large unilamellar vesicles of various compositions. The amount of extracted cholesterol is highly dependent on the composition of lipid membrane, i.e. the presence of sphingomyelin drastically reduced and slowed down cholesterol extraction by methyl-β-cyclodextrin. This was confirmed also in the erythrocyte ghosts system, where more cholesterol was extracted after erythrocytes were treated with sphingomyelinase. We further show that the kinetics of the extraction is mono-exponential for mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol. The kinetics is complex for ternary lipid mixtures composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine, bovine brain sphingomyelin and cholesterol. Our results indicate that the complex kinetics observed in experiments with cells may be the consequence of lateral segregation of lipids in cell plasma membrane.     

Domain nucleation rates and interfacial line tensions in supported bilayers of ternary mixtures containing galactosylceramide

Domains within the plane of the plasma membrane, referred to as membrane rafts, have been a topic of considerable interest in the field of membrane biophysics. Although model membrane systems have been used extensively to study lipid phase behavior as it relates to the existence of rafts, very little work has focused on either the initial stage of lipid domain nucleation, or the relevant physical parameters such as temperature and interfacial line tension which control nucleation. In this work, we utilize a method in which the kinetic process of lipid domain nucleation is imaged by atomic force microscopy and modeled using classical theory of nucleation to map interfacial line tension in ternary lipid mixtures. These mixtures consist of a fluid phase lipid component (1,2-dilauroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, or 1,2-dioleoyl-sn-glycero-3-phosphocholine), a solid phase component (galactosylceramide), and cholesterol. Interfacial line tension measurements of galactosylceramide-rich domains track with our previously measured area/perimeter ratios and height mismatches measured here. Line tension also follows known trends in cholesterol interactions and partitioning, as we observed previously with area/perimeter ratios. Our line tension measurements are discussed in combination with recent line tension measurements to address line tension regulation by cholesterol and the dynamic nature of membrane rafts.     

Specific RNA binding to ordered phospholipid bilayers

We have studied RNA binding to vesicles bounded by ordered and disordered phospholipid membranes. A positive correlation exists between bilayer order and RNA affinity. In particular, structure-dependent RNA binding appears for rafted (liquid-ordered) domains in sphingomyelin-cholesterol-1,2-dioleoyl-sn-glycero-3-phosphocholine vesicles. Binding to more highly ordered gel phase membranes is stronger, but much less RNA structure-dependent. All modes of RNA-membrane association seem to be electrostatic and headgroup directed. Fluorometry on 1,2-dimyristoyl-sn-glycero-3-phosphocholine liposomes indicates that bound RNA broadens the gel-fluid melting transition, and reduces lipid headgroup order, as detected via fluorometric measurement of intramembrane electric fields. RNA preference for rafted lipid was visualized and confirmed using multiple fluorophores that allow fluorescence and fluorescence resonance energy transfer microscopy on RNA molecules closely associated with ordered lipid patches within giant vesicles. Accordingly, both RNA structure and membrane order could modulate biological RNA–membrane interactions.     

Asymmetric structural features in single supported lipid bilayers containing cholesterol and GM1 resolved with synchrotron X-Ray reflectivity

The cell membrane comprises numerous protein and lipid molecules capable of asymmetric organization between leaflets and liquid-liquid phase separation. We use single supported lipid bilayers (SLBs) to model cell membranes, and study how cholesterol and asymmetrically oriented ganglioside receptor G_M1 affect membrane structure using synchrotron x-ray reflectivity. Using mixtures of cholesterol, sphingomyelin, and 1,2-dioleoyl-sn-glycero-3-phosphocholine, we characterize the structure of liquid-ordered and liquid-disordered SLBs in terms of acyl-chain density, headgroup size, and leaflet thickness. SLBs modeling the liquid-ordered phase are 10 Å thicker and have a higher acyl-chain electron density (〈ρ_chain〉 = 0.33 e⁻/ų) compared to SLBs modeling the liquid-disordered phase, or pure phosphatidylcholine SLBs (〈ρ_chain〉 = 0.28 e⁻/ų). Incorporating G_M1 into the distal bilayer leaflet results in membrane asymmetry and thickening of the leaflet of 4-9 Å. The structural effect of G_M1 is more complex in SLBs of cholesterol/sphingomyelin/1,2-dioleoyl-sn-glycero-3-phosphocholine, where the distal chains show a high electron density (〈ρ_chain〉 = 0.33 e⁻/ų) and the lipid diffusion constant is reduced by ∼50%, as measured by fluorescence microscopy. These results give quantitative information about the leaflet asymmetry and electron density changes induced by receptor molecules that penetrate a single lipid bilayer.     

HIV Fusion Peptide Penetrates, Disorders, and Softens T-Cell Membrane Mimics

This work investigates the interaction of N-terminal gp41 fusion peptide (FP) of human immunodeficiency virus type 1 (HIV-1) with model membranes in order to elucidate how FP leads to fusion of HIV and T-cell membranes. FP constructs were (i) wild-type FP23 (23 N-terminal amino acids of gp41), (ii) water-soluble monomeric FP that adds six lysines on the C-terminus of FP23 (FPwsm), and (iii) the C-terminus covalently linked trimeric version (FPtri) of FPwsm. Model membranes were (i) LM3 (a T-cell mimic), (ii) 1,2-dioleoyl-sn-glycero-3-phosphocholine, (iii) 1,2-dioleoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol, (iv) 1,2-dierucoyl-sn-glycero-3-phosphocholine, and (v) 1,2-dierucoyl-sn-glycero-3-phosphocholine/30 mol% cholesterol. Diffuse synchrotron low-angle x-ray scattering from fully hydrated samples, supplemented by volumetric data, showed that FP23 and FPtri penetrate into the hydrocarbon region and cause membranes to thin. Depth of penetration appears to depend upon a complex combination of factors including bilayer thickness, presence of cholesterol, and electrostatics. X-ray data showed an increase in curvature in hexagonal phase 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, which further indicates that FP23 penetrates into the hydrocarbon region rather than residing in the interfacial headgroup region. Low-angle x-ray scattering data also yielded the bending modulus K_C, a measure of membrane stiffness, and wide-angle x-ray scattering yielded the S_xray orientational order parameter. Both FP23 and FPtri decreased K_C and S_xray considerably, while the weak effect of FPwsm suggests that it did not partition strongly into LM3 model membranes. Our results are consistent with the HIV FP disordering and softening the T-cell membrane, thereby lowering the activation energy for viral membrane fusion.     

Structure and Dynamics of the Myristoyl Lipid Modification of Src Peptides Determined by H Solid-State NMR Spectroscopy

Lipid modifications of proteins are widespread in nature and play an important role in numerous biological processes. The nonreceptor tyrosine kinase Src is equipped with an N-terminal myristoyl chain and a cluster of basic amino acids for the stable membrane association of the protein. We used ²H NMR spectroscopy to investigate the structure and dynamics of the myristoyl chain of myr-Src(2-19), and compare them with the hydrocarbon chains of the surrounding phospholipids in bilayers of varying surface potentials and chain lengths. The myristoyl chain of Src was well inserted in all bilayers investigated. In zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine membranes, the myristoyl chain of Src was significantly longer and appears “stiffer” than the phospholipid chains. This can be explained by an equilibrium between the attraction attributable to the insertion of the myristoyl chain and the Born repulsion. In a 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-[phospho-L-serine] membrane, where attractive electrostatic interactions come into play, the differences between the peptide and the phospholipid chain lengths were attenuated, and the molecular dynamics of all lipid chains were similar. In a much thicker 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-[phospho-L-serine]/cholesterol membrane, the length of the myristoyl chain of Src was elongated nearly to its maximum, and the order parameters of the Src chain were comparable to those of the surrounding membrane.     

Morphometric Image Analysis of Giant Vesicles: A New Tool for Quantitative Thermodynamics Studies of Phase Separation in Lipid Membranes

We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3-phosphocholine/cholesterol. In general, our results show a reasonable agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence-microscopy experiments in GUVs, particularly retrieving quantitative data for the construction of three lipid-component-phase diagrams containing cholesterol.     

FRET Detects the Size of Nanodomains for Coexisting Liquid-Disordered and Liquid-Ordered Phases

Biomembranes with as few as three lipid components can form coexisting liquid-disordered (Ld) and liquid-ordered (Lo) phases. In the coexistence region of Ld and Lo phases, the lipid mixtures 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/chol or brain sphingomyelin (bSM)/DOPC/chol form micron-scale domains that are easily visualized with light microscopy. Although large domains are not observed in the mixtures DSPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/chol and bSM/POPC/chol, lateral heterogeneity is nevertheless detected using techniques with nanometer-scale spatial resolution. We propose a simple and accessible method to measure domain sizes below optical resolution (～200 nm). We measured nanodomain size for the latter two mixtures by combining experimental Förster resonance energy transfer data with a Monte-Carlo-based analysis. We found a domain radius of 7.5?10 nm for DSPC/POPC/chol, similar to values obtained previously by neutron scattering, and ～5 nm for bSM/POPC/chol, slightly smaller than measurable by neutron scattering. These analyses also detect the domain-size transition that is observed by fluorescence microscopy in the four-component lipid mixture bSM/DOPC/POPC/chol. Accurate measurements of fluorescent-probe partition coefficients are especially important for the analysis; therefore, we exploit three different methods to measure the partition coefficient of fluorescent molecules between Ld and Lo phases.     

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.     

N-Nervonoylsphingomyelin (C24:1) Prevents Lateral Heterogeneity in Cholesterol-Containing Membranes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.     

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2–10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by ³¹P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2–3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.     

Development of fluorophore dynamics imaging as a probe for lipid domains in model vesicles and cell membranes

The ability to detect raft structures in membranes continues to present a problem, especially in the membranes of live cells. Rafts, generally considered to be small (<200 nm) sphingolipid-rich regions, are commonly modelled using lipid vesicle systems where the ability of fluorophore-labelled lipids to preferentially locate into domains (basically large rafts) is investigated. Instead, in this study the motional properties of different fluorophores were determined using two-photon excitation and time-correlated single-photon counting coupled with diffraction-limited imaging with polarizing optics in scanning mode to obtain nanosecond rotational correlation time images. To develop the method, well-characterized domain-containing models consisting of giant unilamellar vesicles comprising mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin and cholesterol were used with the fluorophores diphenylhexatriene, 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl). Accordingly, images of rotational correlation times of the probes revealed domain structures for all three probes consistent with other studies using different approaches. Rotational correlation time images of living cell membranes were also observed. The method has the advantage that not only does it enable domains to be visualised or imaged in a unique manner but that it can also potentially provide useful information on the lipid dynamics within the structures.     

Characterization of the binary mixed monolayers of α-tocopherol with phospholipids at the air-water interface

The mixed Langmuir monolayers composed of model constituents of biological membranes, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), were investigated to provide information on the intermolecular interactions between these membrane components and the physiologically active vitamin E–α-tocopherol (TF), as well as on the phase behavior of these mixed systems. Additionally, topography of these monolayers transferred onto the mica support was investigated by the inverted metallurgical microscope. Morphological characteristics were directly observed by Brewster angle microscopy (BAM). From the surface pressure–area isotherms and the analysis of physicochemical parameters (compressibility and mean molecular area at the maximum compressibility) it was found that depending on the acyl chains saturation degree, TF has different effect on the phospholipids. In the case of DPPC, the addition of TF to the phospholipid film causes destabilization of the ordered hydrocarbon chains, while in the POPC/DOPC–TF systems, the attractive interactions are responsible for the monolayer increased stability. Thus, the results of these studies confirm the hypothesis that α-tocopherol may play a role in the stabilization of biological membranes.     

Liquid-liquid domains in bilayers detected by wide angle X-ray scattering

Wide angle x-ray scattering (WAXS) from oriented lipid multilayers is used to examine liquid-ordered (Lo)/liquid-disordered (Ld) phase coexistence in the system 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DOPC/DPPC/Chol), which is a model for the outer leaflet of the animal cell plasma membrane. Using the method of analysis developed in the accompanying work, we find that two orientational distributions are necessary to fit the WAXS data at lower temperatures, whereas only one distribution is needed at temperatures higher than the miscibility transition temperature, T_mix = 25-35°C (for 1:1 DOPC/DPPC with 15%, 20%, 25%, and 30% Chol). We propose that the necessity for two distributions is a criterion for coexistence of Lo domains with a high S_x-ray order parameter and Ld domains with a lower order parameter. This criterion is capable of detecting coexistence of small domains or rafts that the conventional x-ray criterion of two lamellar D spacings may not. Our T_mix values tend to be slightly larger than published NMR results and microscopy results when the fluorescence probe artifact is considered. This is consistent with the sensitivity of WAXS to very short time and length scales, which makes it more capable of detecting small, short-lived domains that are likely close to T_mix.     

Cholesterol Partition and Condensing Effect in Phase-Separated Ternary Mixture Lipid Multilayers

The cholesterol partitioning and condensing effect in the liquid-ordered (L_o) and liquid-disordered (L_d) phases were systematically investigated for ternary mixture lipid multilayers consisting of 1:1 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine with varying concentrations of cholesterol. X-ray lamellar diffraction was used to deduce the electron density profiles of each phase. The cholesterol concentration in each phase was quantified by fitting of the electron density profiles with a newly invented basic lipid profile scaling method that minimizes the number of fitting parameters. The obtained cholesterol concentration in each phase versus total cholesterol concentration in the sample increases linearly for both phases. The condensing effect of cholesterol in ternary lipid mixtures was evaluated in terms of phosphate-to-phosphate distances, which together with the estimated cholesterol concentration in each phase was converted into an average area per molecule. In addition, the cholesterol position was determined to a precision of (±0.7Å) and an increase of disorder in the lipid packing in the L_o phase was observed for total cholesterol concentration of 20∼30%.     

Lipid Reorganization Induced by Shiga Toxin Clustering on Planar Membranes

The homopentameric B-subunit of bacterial protein Shiga toxin (STxB) binds to the glycolipid Gb₃ in plasma membranes, which is the initial step for entering cells by a clathrin-independent mechanism. It has been suggested that protein clustering and lipid reorganization determine toxin uptake into cells. Here, we elucidated the molecular requirements for STxB induced Gb₃ clustering and for the proposed lipid reorganization in planar membranes. The influence of binding site III of the B-subunit as well as the Gb₃ lipid structure was investigated by means of high resolution methods such as fluorescence and scanning force microscopy. STxB was found to form protein clusters on homogenous 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol/Gb₃ (65∶30∶5) bilayers. In contrast, membranes composed of DOPC/cholesterol/sphingomyelin/Gb₃ (40∶35∶20∶5) phase separate into a liquid ordered and liquid disordered phase. Dependent on the fatty acid composition of Gb₃, STxB-Gb₃ complexes organize within the liquid ordered phase upon protein binding. Our findings suggest that STxB is capable of forming a new membrane phase that is characterized by lipid compaction. The significance of this finding is discussed in the context of Shiga toxin-induced formation of endocytic membrane invaginations.     

Partitioning of Tetrachlorophenol into Lipid Bilayers and Sarcoplasmic Reticulum: Effect of Length of Acyl Chains,Carbonyl Group of Lipids and Biomembrane Structure

We report results of a partitioning study of 2,3,4,6-tetrachlorophenol (TeCP). In the study we explored (1) the effect of the length of acyl chains of lipids (C16:1 – C24:1) and alkanes (C6–C16), (2) the role of the carbonyl group of lipids, and (3) the effect of molecular structure of the sarcoplasmic reticulum membrane on TeCP partitioning. Mole fraction partition coefficients have been measured using equilibrium dialysis for un-ionized (HA), and ionized (A) species, Kp_x^HA, Kp_x^A. Their values are concentration-dependent. Partition coefficients were analyzed in terms of a model that accounts for saturation of membrane associated with the finite area of partition site, and electrostatic interactions of (A-) species with charged membrane. Limiting values of partition coefficients, corresponding to infinite dilution of solute, Kp_x0^HA, Kp_x0^A were obtained. Kp_x0^HA and Kp_x0^A measure the strength of solute-membrane interactions. Studies were done with single-layered vesicles of lipids with variable chain length: 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (C16:1), 1,2-dioleoyl-sn-glycero-3-phosphocholine (C18:1), 1,2-dierucoyl-sn-glycero-3-phosphocholine (C22:1), and 1 ,2-dinervonoyl-sn-glycero-3-phosphocholine (C24:1), and egg-PC. Kp_x0 for transfer of TeCP from water into lipid membranes was found to be independent of the length of acyl chains, whereas Kp_x0 for transfer from water into alkanes increased with the length of alkane. The effect of the carbonyl CO group of lipids on partitioning was measured using 1,2-di-o-octadecenyl-sn-glycero-3-phosphocholine (CO absent) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (CO present) liposomes. Carbonyl groups, known to change dipolar potential, had no effect on partitioning. Partition coefficients of un-ionized and ionized forms of TeCP were invariant to the presence of proteins and other membrane components of sarcoplasmic reticulum (SR) membrane.     

Order parameters and areas in fluid-phase oriented lipid membranes using wide angle X-ray scattering

We used wide angle x-ray scattering (WAXS) from stacks of oriented lipid bilayers to measure chain orientational order parameters and lipid areas in model membranes consisting of mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/cholesterol in fluid phases. The addition of 40% cholesterol to either DOPC or DPPC changes the WAXS pattern due to an increase in acyl chain orientational order, which is one of the main properties distinguishing the cholesterol-rich liquid-ordered (Lo) phase from the liquid-disordered (Ld) phase. In contrast, powder x-ray data from multilamellar vesicles does not yield information about orientational order, and the scattering from the Lo and Ld phases looks similar. An analytical model to describe the relationship between the chain orientational distribution and WAXS data was used to obtain an average orientational order parameter, S_x-ray. When 40% cholesterol is added to either DOPC or DPPC, S_x-ray more than doubles, consistent with previous NMR order parameter measurements. By combining information about the average chain orientation with the chain-chain correlation spacing, we extended a commonly used method for calculating areas for gel-phase lipids to fluid-phase lipids and obtained agreement to within 5% of literature values.     

Oxygen distribution in the fluid/gel phases of lipid membranes

The interactions between oxygen and lipid membranes play fundamental roles in basic biological processes (e.g., cellular respiration). Obviously, membrane oxidation is expected to be critically dependent on the distribution and concentration of oxygen in the membrane. Here, we combined theoretical and experimental methods to investigate oxygen partition and distribution in lipid membranes of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a temperature range between 298 and 323 K, specifically focusing on the changes caused by the lipid phase and phase transition. Even though oxygen is known to be more concentrated in the center of fluid phase membranes than on the headgroup regions, the distribution profile of oxygen inside gel-phase bilayers remained to be determined. Molecular dynamics simulations now show that the distribution of oxygen inside DPPC bilayers dramatically changes upon crossing the main transition temperature, with oxygen being nearly depleted halfway from the headgroups to the membrane center below the transition temperature. In a parallel approach, singlet oxygen luminescence emission measurements employing the photosensitizer Pheophorbide-a (Pheo) confirmed the differences in oxygen distribution and concentration profiles between gel- and fluid-phase membranes, revealing changes in the microenvironment of the embedded photosensitizer. Our results also reveal that excited triplet state lifetime, as it can be determined from the singlet oxygen luminescence kinetics, is a useful probe to assess oxygen distribution in lipid membranes with distinct lipid compositions.