Cholesterol In Bilayers Research Articles

Probing the Origins of the Disorder-to-Order Transition of a Modified Cholesterol in Ternary Lipid Bilayers.

In a recent study, spectroscopic observations of modified cholesterol in both lipid-coated nanoparticles and liposomes provided evidence for a disorder-to-order orientational transition with increasing temperature. Below a critical temperature, in a membrane composed of modified cholesterol, saturated (DPPC) lipid, and anionic (DOPS) lipid, a roughly equal population of head-out and head-in conformations was observed. Surprisingly, as temperature was increased the modified cholesterol presented an abrupt transition to a population of all head-in orientations. Additionally, when saturated DPPC lipids were replaced by unsaturated DOPC the disorder-to-order transition was eliminated. To gain insight into this curious transition, we use all-atom molecular dynamics simulations to characterize the structure and fluctuations of lipid bilayers composed of saturated and unsaturated lipids, in the presence of normal and modified cholesterol. Free energy differences between head-out and head-in conformations are computed as a function of varying lipid membrane composition for normal and modified cholesterol. In bilayers primarily composed of DPPC, the orientation of modified cholesterol is observed to depend sensitively on the orientation of the surrounding normal or modified cholesterol molecules, suggesting cooperative Ising-like interactions favoring an ordered state. In bilayers primarily composed of DOPC, spontaneous flip-flop of modified cholesterol is observed, consistent with the measured small free energy barrier separating the head-in and head-out orientations. This combined experimental and computational study effectively characterizes the orientational dimorphism and provides novel insight into the fundamental nature of cholesterol interactions in membrane.

Influence of Simvastatin and Pravastatin on the Biophysical Properties of Model Lipid Bilayers and Plasma Membranes of Live Cells.

Statins are among the most widely used drugs for the inhibition of cholesterol biosynthesis, prevention of cardiovascular diseases, and treatment of hypercholesterolemia. Additionally, statins also exhibit cholesterol-independent benefits in various diseases, including neuroprotective properties in Alzheimer's disease, anti-inflammatory effects in coronary artery disease, and antiproliferative activities in cancer, which likely result from the statins' interaction and alteration of lipid bilayers. However, the membrane-modulatory effects of statins and the mechanisms by which statins alter lipid bilayers remain poorly understood. In this work, we explore the membrane-modulating effects of statins on model lipid bilayers and live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM) combined with viscosity-sensitive environmental probes, we demonstrate that hydrophobic, but not hydrophilic, statins are capable of changing the microviscosity and lipid order in model and live cell membranes. Furthermore, we show that hydrophobic simvastatin is capable of forming nanoscale cholesterol-rich domains and homogenizing the cholesterol concentrations in lipid bilayers. Our results provide a mechanistic framework for understanding the bimodal effects of simvastatin on the lipid order and the lateral organization of cholesterol in lipid bilayers. Finally, we demonstrate that simvastatin temporarily decreases the microviscosity of live cell plasma membranes, making them more permeable and increasing the level of intracellular chemotherapeutic drug accumulation.

Molecular Insights into the Effect of Cholesterol on the Binding of Bicarbonate Ions in Band 3 Protein.

Band 3, or anion exchanger 1 (AE1), is one of the indispensable transmembrane proteins involved in the effective respiratory process of the human body and is primarily responsible for the exchange of bicarbonate and chloride anions across the plasma membrane of erythrocyte. However, the molecular mechanism of ion transport of Band 3 is not completely understood, yet. In this work, we systematically investigate the key binding sites of bicarbonate ions in Band 3 and the impact of cholesterol (CHOL) in lipid bilayers on bicarbonate ion binding using all-atom molecular dynamics (MD) simulations. We examine the dynamics of interactions of bicarbonate ions with Band 3 in the microsecond time scale and calculate the binding free energy of the anion in Band 3. The results indicate that the residue R730 of Band 3 is the most probable binding site for bicarbonate ions. CHOL enhances the bicarbonate ion binding by influencing the conformational stability of Band 3 and compressing the volume of the Band 3 cavity. These findings provide some insights into the bicarbonate ion binding in Band 3 and are helpful for understanding the anion exchange of Band 3.

Cholesterol Content Regulates the Interaction of αA-, αB-, and α-Crystallin with the Model of Human Lens-Lipid Membranes.

α-Crystallin (αABc) is a major protein comprised of αA-crystallin (αAc) and αB-crystallin (αBc) that is found in the human eye lens and works as a molecular chaperone by preventing the aggregation of proteins and providing tolerance to stress. However, with age and cataract formation, the concentration of αABc in the eye lens cytoplasm decreases, with a corresponding increase in the membrane-bound αABc. This study uses the electron paramagnetic resonance (EPR) spin-labeling method to investigate the role of cholesterol (Chol) and Chol bilayer domains (CBDs) in the binding of αAc, αBc, and αABc to the Chol/model of human lens-lipid (Chol/MHLL) membranes. The maximum percentage of membrane surface occupied (MMSO) by αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trends: MMSO (αAc) > MMSO (αBc) ≈ MMSO (αABc), indicating that a higher amount of αAc binds to these membranes compared to αBc and αABc. However, with an increase in the Chol concentration in the Chol/MHLL membranes, the MMSO by αAc, αBc, and αABc decreases until it is completely diminished at a mixing ratio of 1.5. The Ka of αAc, αBc, and αABc to Chol/MHLL membranes at a mixing ratio of 0 followed the trend: Ka (αBc) ≈ Ka (αABc) > Ka (αAc), but it was close to zero with the diminished binding at a Chol/MHLL mixing ratio of 1.5. The mobility near the membrane headgroup regions decreased with αAc, αBc, and αABc binding, and the Chol antagonized the capacity of the αAc, αBc, and αABc to decrease mobility near the headgroup regions. No significant change in membrane order near the headgroup regions was observed, with an increase in αAc, αBc, and αABc concentrations. Our results show that αAc, αBc, and αABc bind differently with Chol/MHLL membranes at mixing ratios of 0 and 0.5, decreasing the mobility and increasing hydrophobicity near the membrane headgroup region, likely forming the hydrophobic barrier for the passage of polar and ionic molecules, including antioxidants (glutathione), creating an oxidative environment inside the lens, leading to the development of cataracts. However, all binding was completely diminished at a mixing ratio of 1.5, indicating that high Chol and CBDs inhibit the binding of αAc, αBc, and αABc to membranes, preventing the formation of hydrophobic barriers and likely protecting against cataract formation.

Association of Alpha-Crystallin with Human Cortical and Nuclear Lens Lipid Membrane Increases with the Grade of Cortical and Nuclear Cataract.

Eye lens α-crystallin has been shown to become increasingly membrane-bound with age and cataract formation; however, to our knowledge, no studies have investigated the membrane interactions of α-crystallin throughout the development of cataracts in separated cortical membrane (CM) and nuclear membrane (NM) from single human lenses. In this study, four pairs of human lenses from age-matched male and female donors and one pair of male lenses ranging in age from 64 to 73 years old (yo) were obtained to investigate the interactions of α-crystallin with the NM and CM throughout the progression of cortical cataract (CC) and nuclear cataract (NC) using the electron paramagnetic resonance spin-labeling method. Donor health history information (diabetes, smoker, hypertension, radiation treatment), sex, and race were included in the data analysis. The right eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 1, NC: 2), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Similarly, left eye lenses CM and NM investigated were 64 yo male (CC: 0), 68 yo male (CC: 3, NC: 2), 73 yo male (CC: 2, NC: 3), 68 yo female (CC: 3, NC: 2), and 73 yo female (CC: 1, NC: 3). Analysis of α-crystallin binding to male and female eye lens CM and NM revealed that the percentage of membrane surface occupied (MSO) by α-crystallin increases with increasing grade of CC and NC. The binding of α-crystallin resulted in decreased mobility, increased order, and increased hydrophobicity on the membrane surface in male and female eye lens CM and NM. CM mobility decreased with an increase in cataracts for both males and females, whereas the male lens NM mobility showed no significant change, while female lens NM showed increased mobility with an increase in cataract grade. Our data shows that a 68 yo female donor (long-term smoker, pre-diabetic, and hypertension; grade 3 CC) showed the largest MSO by α-crystallin in CM from both the left and right lens and had the most pronounced mobility changes relative to all other analyzed samples. The variation in cholesterol (Chol) content, size and amount of cholesterol bilayer domains (CBDs), and lipid composition in the CM and NM with age and cataract might result in a variation of membrane surface mobility, membrane surface hydrophobicity, and the interactions of α-crystallin at the surface of each CM and NM. These findings provide insight into the effect of decreased Chol content and the reduced size and amount of CBDs in the cataractous CM and NM with an increased binding of α-crystallin with increased CC and NC grade, which suggests that Chol and CBDs might be a key component in maintaining lens transparency.

Insight into molecular structures and dynamical properties of niosome bilayers containing melatonin molecules: a molecular dynamics simulation approach.

Niosomes represent vesicular carriers capable of encapsulating both hydrophobic and hydrophilic drugs within their inner core or bilayer shell. They are typically composed of non-ionic synthetic surfactants such as sorbitan monostearate (Span60) with the addition of cholesterol (Chol). The physical properties and stability of niosomal vesicles strongly depend on the composition of their bilayers, which plays a significant role in determining the efficiency of drug encapsulation and release in drug delivery systems. In this study, we have explored the interactions between melatonin (Mel) molecules and the niosome bilayer, as well as their resulting physical properties. Molecular dynamics simulations were employed to investigate melatonin-inserted niosome bilayers, both with and without the inclusion of cholesterol. The simulation results revealed that cholesterol notably influences the location of melatonin molecules within the niosome bilayers. In the absence of cholesterol, melatonin tends to occupy the region around the Span60 tail groups. However, in the presence of cholesterol, melatonin is found in the vicinity of the Span60 head groups. Melatonin molecules in niosome bilayers without cholesterol exhibit a more ordered orientation when compared to those in bilayers containing 50 mol% cholesterol. The bilayer structure of the Span60/Mel and Span60/Chol/Mel systems exhibited a liquid-disordered phase (Ld). In contrast, the Span60/Chol bilayer system displays a liquid-ordered phase (Lo) with less fluidity. This study reveals that melatonin induces a disorderly bilayer structure and greater lateral expansion, whereas cholesterol induces an orderly bilayer structure and a more condensed effect. Cholesterol plays a crucial role in condensing the bilayer structure with stronger interactions between Span60 and cholesterol. The addition of 50 mol% cholesterol in the Span60 bilayers not only enhances the stability and rigidity of niosomes but also facilitates the easier release of melatonin from the bilayer membranes. This finding is particularly valuable in the context of preparing niosomes for drug delivery systems.

QS21-Initiated Fusion of Liposomal Small Unilamellar Vesicles to Form ALFQ Results in Concentration of Most of the Monophosphoryl Lipid A, QS21, and Cholesterol in Giant Unilamellar Vesicles.

Army Liposome Formulation with QS21 (ALFQ), a vaccine adjuvant preparation, comprises liposomes containing saturated phospholipids, with 55 mol% cholesterol relative to the phospholipids, and two adjuvants, monophosphoryl lipid A (MPLA) and QS21 saponin. A unique feature of ALFQ is the formation of giant unilamellar vesicles (GUVs) having diameters >1.0 µm, due to a remarkable fusion event initiated during the addition of QS21 to nanoliposomes containing MPLA and 55 mol% cholesterol relative to the total phospholipids. This results in a polydisperse size distribution of ALFQ particles, with diameters ranging from ~50 nm to ~30,000 nm. The purpose of this work was to gain insights into the unique fusion reaction of nanovesicles leading to GUVs induced by QS21. This fusion reaction was probed by comparing the lipid compositions and structures of vesicles purified from ALFQ, which were >1 µm (i.e., GUVs) and the smaller vesicles with diameter <1 µm. Here, we demonstrate that after differential centrifugation, cholesterol, MPLA, and QS21 in the liposomal phospholipid bilayers were present mainly in GUVs (in the pellet). Presumably, this occurred by rapid lateral diffusion during the transition from nanosize to microsize particles. While liposomal phospholipid recoveries by weight in the pellet and supernatant were 44% and 36%, respectively, higher percentages by weight of the cholesterol (~88%), MPLA (94%), and QS21 (96%) were recovered in the pellet containing GUVs, and ≤10% of these individual liposomal constituents were recovered in the supernatant. Despite the polydispersity of ALFQ, most of the cholesterol, and almost all of the adjuvant molecules, were present in the GUVs. We hypothesize that the binding of QS21 to cholesterol caused new structural nanodomains, and subsequent interleaflet coupling in the lipid bilayer might have initiated the fusion process, leading to creation of GUVs. However, the polar regions of MPLA and QS21 together have a "sugar lawn" of ten sugars, the hydrophilicity of which might have provided a driving force for rapid lateral diffusion and concentration of the MPLA and QS21 in the GUVs.

Graph-Based Analyses of Dynamic Water-Mediated Hydrogen-Bond Networks in Phosphatidylserine: Cholesterol Membranes.

Phosphatidylserine lipids are anionic molecules present in eukaryotic plasma membranes, where they have essential physiological roles. The altered distribution of phosphatidylserine in cells such as apoptotic cancer cells, which, unlike healthy cells, expose phosphatidylserine, is of direct interest for the development of biomarkers. We present here applications of a recently implemented Depth-First-Search graph algorithm to dissect the dynamics of transient water-mediated lipid clusters at the interface of a model bilayer composed of 1-palmytoyl-2-oleoyl-sn-glycero-2-phosphatidylserine (POPS) and cholesterol. Relative to a reference POPS bilayer without cholesterol, in the POPS:cholesterol bilayer there is a somewhat less frequent sampling of relatively complex and extended water-mediated hydrogen-bond networks of POPS headgroups. The analysis protocol used here is more generally applicable to other lipid:cholesterol bilayers.

Real-time tracking of drug binding to influenza A M2 reveals a high energy barrier

The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.

Cholesterol Biases the Conformational Landscape of the Chemokine Receptor CCR3: A MAS SSNMR-Filtered Molecular Dynamics Study.

Cholesterol directs the pathway of ligand-induced G protein-coupled receptor (GPCR) signal transduction. The GPCR C-C motif chemokine receptor 3 (CCR3) is the principal chemotactic receptor for eosinophils, with roles in cancer metastasis and autoinflammatory conditions. Recently, we discovered a direct correlation between bilayer cholesterol and increased agonist-triggered CCR3 signal transduction. However, the allosteric molecular mechanism escalating ligand affinity and G protein coupling is unknown. To study cholesterol-guided CCR3 conformational selection, we implement comparative, objective measurement of protein architectures by scoring shifts (COMPASS) to grade model structures from molecular dynamics simulations. In this workflow, we scored predicted chemical shifts against 2-dimensional solid-state NMR 13C-13C correlation spectra of U-15N,13C-CCR3 samples prepared with and without cholesterol. Our analysis of trajectory model structures uncovers that cholesterol induces site-specific conformational restraint of extracellular loop (ECL) 2 and conserved motion in transmembrane helices and ECL3 not observed in simulations of bilayers with only phosphatidylcholine lipids. PyLipID analysis implicates direct cholesterol agency in CCR3 conformational selection and dynamics. Residue-residue contact scoring shows that cholesterol biases the conformational selection of the orthosteric pocket involving Y411.39, Y1133.32, and E2877.39. Lastly, we observe contact remodeling in activation pathway residues centered on the initial transmission switch, Na+ pocket, and R3.50 in the DRY motif. Our observations have unique implications for understanding of CCR3 ligand recognition and specificity and provide mechanistic insight into how cholesterol functions as an allosteric regulator of CCR3 signal transduction.

Interdependence of cholesterol distribution and conformational order in lipid bilayers.

We show, via molecular simulations, that not only does cholesterol induce a lipid order, but the lipid order also enhances cholesterol localization within the lipid leaflets. Therefore, there is a strong interdependence between these two phenomena. In the ordered phase, cholesterol molecules are predominantly present in the bilayer leaflets and orient themselves parallel to the bilayer normal. In the disordered phase, cholesterol molecules are mainly present near the center of the bilayer at the midplane region and are oriented orthogonal to the bilayer normal. At the melting temperature of the lipid bilayers, cholesterol concentration in the leaflets and the bilayer midplane is equal. This result suggests that the localization of cholesterol in the lipid bilayers is mainly dictated by the degree of ordering of the lipid bilayer. We validate our findings on 18 different lipid bilayer systems, obtained from three different phospholipid bilayers with varying concentrations of cholesterol. To cover a large temperature range in simulations, we employ the Dry Martini force field. We demonstrate that the Dry and the Wet Martini (with polarizable water) force fields produce comparable results.

Cholesterol solubility in mixed DMPE/DMPC bilayers as determined by small angle X-ray scattering

Small angle X-ray scattering measurements under ambient conditions (T ≈ 294 K) provide evidence for the formation of separate domains in a ternary, mixed phospholipid ([DMPE]/[DMPC] = 3/1) / cholesterol model bilayer membrane. As we interpret these results, the domains contain cholesterol and DMPC, with which cholesterol is known to preferentially interact in a binary model membrane (solubility limit, mol fraction cholesterol 0.5), as compared to DMPE (solubility limit, mol fraction cholesterol 0.45). The solubility limit for the ternary system is mol fraction cholesterol 0.2–0.3. Although literature EPR spectra find that non-crystalline, cholesterol bilayer domains may be present even prior to the observation of cholesterol crystal diffraction, X-ray scattering cannot detect their presence.

Membrane Models and Experiments Suitable for Studies of the Cholesterol Bilayer Domains.

Cholesterol (Chol) is an essential component of animal cell membranes and is most abundant in plasma membranes (PMs) where its concentration typically ranges from 10 to 30 mol%. However, in red blood cells and Schwann cells, PMs Chol content is as high as 50 mol%, and in the PMs of the eye lens fiber cells, it can reach up to 66 mol%. Being amphiphilic, Chol molecules are easily incorporated into the lipid bilayer where they affect the membrane lateral organization and transmembrane physical properties. In the aqueous phase, Chol cannot form free bilayers by itself. However, pure Chol bilayer domains (CBDs) can form in lipid bilayer membranes with the Chol content exceeding 50 mol%. The range of Chol concentrations surpassing 50 mol% is less frequent in biological membranes and is consequently less investigated. Nevertheless, it is significant for the normal functioning of the eye lens and understanding how Chol plaques form in atherosclerosis. The most commonly used membrane models are unilamellar and multilamellar vesicles (MLVs) and supported lipid bilayers (SLBs). CBDs have been observed directly using confocal microscopy, X-ray reflectometry and saturation recovery electron paramagnetic resonance (SR EPR). Indirect evidence of CBDs has also been reported by using atomic force microscopy (AFM) and fluorescence recovery after photobleaching (FRAP) experiments. The overall goal of this review is to demonstrate the advantages and limitations of the various membrane models and experimental techniques suitable for the detection and investigation of the lateral organization, function and physical properties of CBDs.

The Effect of Cholesterol in SOPC Lipid Bilayers at Low Temperatures.

We study the behavior of lipid bilayers composed of SOPC (1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine) with different concentrations of cholesterol, ranging from 10 mol% to 50 mol% at 273 K. To this end, we carry out extensive atomistic molecular dynamic simulations with the aid of the Slipid force field aiming at computing basic bilayer parameters, as well as thermodynamic properties and structural characteristics. The obtained results are compared to available relevant experimental data and the outcome of atomistic simulations performed on bilayers composed of analogous phospholipids. Our results show a good quantitative, as well as qualitative, agreement with the main trends associated with the concentration increase in cholesterol. Moreover, it comes out that a change in the behavior of the bilayer is brought about at a concentration of about 30 mol% cholesterol. At this very concentration, some of the bilayer properties are found to exhibit a saturation and a significant long-range ordering of the lipid molecules in the membrane shows up.

Cholesterol concentration effect on bilayer membranes and its role in designing efficient liposomal drug delivery systems.

Liposomal drug delivery systems provide a versatile therapeutic platform for treating numerous diseases. Cholesterol, as a common component of many liposomal drug delivery systems, plays a crucial role in enhancing mechanical strength and decreasing the permeability of biomembranes. However, the ratio required for a proper formulation is poorly recognized. In this study, all-atom molecular dynamics simulations are performed to characterize the effect of cholesterol concentration in bilayers. To prepare liposomes, we simulate DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) phospholipid combined with different molar ratios of cholesterol (100, 80-20, 70-30, and 60-40%) in both planar and spherical bilayers. Furthermore, it has been quantified how a range of bilayer properties, including area per lipid, leaflet interdigitation, membrane thickness, and lipid Scd order parameters, are altered by variation in concentrations of lipid and cholesterol. Therefore, the most suitable amount of cholesterol and lipid to prepare stable and controlled drug release vehicles is obtained by screening the lipid and cholesterol ratio arrangement. Compared to membranes without cholesterol, the influence of cholesterol on lipid bilayers is revealed. These results provide a better understanding of the fundamental characteristics of the structure and dynamics of cholesterol-containing membranes. By revealing molecular details of interactions between cholesterol and phospholipids, these simulations accurately represent atomic-level characteristics of the planar and spherical bilayers. The simulations presented here shed light on the workings and limitations of liposomal drug delivery technology and establish a computational framework for the rational design of liposomes in drug delivery systems.

A coarse-grained molecular dynamics investigation on spontaneous binding of Aβ1–40 fibrils with cholesterol-mixed DPPC bilayers

Alzheimer's disease is the most common form of dementia. Its aetiology is characterized by the misfolding and aggregation of amyloid-β (Aβ) peptides into β-sheet-rich Aβ oligomers/fibrils. Although multiple experimental studies have suggested that Aβ oligomers/fibrils interact with the cell membranes and perturb their structures and dynamics, the molecular mechanism of this interaction is still not fully understood. In the present work, we have performed a total of 120 μs-long simulations to investigate the interaction between trimeric or hexameric Aβ1–40 fibrils with either a 100% DPPC bilayer, a 70% DPPC-30% cholesterol bilayer or a 50% DPPC-50% cholesterol bilayer. Our simulation data capture the spontaneous binding of the aqueous Aβ1–40 fibrils with the membranes and show that the central hydrophobic amino acid cluster, the lysine residue adjacent to it and the C-terminal hydrophobic residues are all involved in the process. Moreover, our data show that while the Aβ1–40 fibril does not bind to the 100% DPPC bilayer, its binding affinity for the membrane increases with the amount of cholesterol. Overall, our data suggest that two clusters of hydrophobic residues and one lysine help Aβ1–40 fibrils establish stable interactions with a cholesterol-rich DPPC bilayer. These residues are likely to represent potential target regions for the design of inhibitors, thus opening new avenues in structure-based drug design against Aβ oligomer/fibril-membrane interaction.

Orientation of Cholesterol in Polyunsaturated Lipid Bilayers.

The local normal to the fluid liquid crystalline phase of the lipid membrane is an axis of motional symmetry for the molecules that make up the bilayer. The presence of cholesterol in the membrane increases not only the lipid hydrocarbon chain order but also the strength of the membrane's orienting potential. Cholesterol undergoes rapid reorientation about a diffusion axis that is roughly aligned with the long molecular axis, but there is also a slower reorientation of the diffusion axis, or "wobble", relative to the local bilayer normal. The extent of this second, slower motion depends on the degree of order of the lipids that make up the bilayer. We use 2H nuclear magnetic resonance of deuterium-labeled cholesterol to investigate quantitatively the effect of lipid chain unsaturation on cholesterol orientation in a series of phospholipid bilayers. We find that the hydrocarbon chains in membranes composed of polyunsaturated lipids are much more highly disordered than those in membranes composed of saturated lipids but that cholesterol remains aligned roughly along the bilayer normal.

Extent of raft composition in a model plasma membrane

“Rafts” are nanometer-size inhomogeneities in the plasma membrane that, in the outer leaflet, are enriched in sphingomyelin and cholesterol. They are thought to provide a platform for proteins to carry out biological processes. Here, we employ a model asymmetric plasma membrane to address the question of the range of sphingomyelin and cholesterol compositions in which one would expect the formation of rafts. We define a weight for the likelihood of raft formation and evaluate it as a function of the sphingomyelin mole fraction in the outer leaflet for three bilayers with total cholesterol mole fractions of 0.30, 0.40, and 0.50. Not surprisingly, the weight decreases when there is little sphingomyelin. Less expected, we find that the weight also decreases when there is a large mole fraction of sphingomyelin. The weight is largest in the bilayer with a total cholesterol mole fraction of 0.30 and decreases rapidly with increasing total cholesterol. We explicate the reasons for these behaviors. In the 0.30 cholesterol bilayer, the largest weight occurs at a sphingomyelin mole fraction in the outer leaflet of approximately 0.23. The weight falls to one half its maximum value at sphingomyelin mole fractions of 0.15 and 0.33. In terms of the sphingomyelin mole fraction of the asymmetric bilayer, the maximum weight occurs at 0.12 and falls to half maximum at 0.08 and 0.17.

Cholesterol Protects the Liquid-Ordered Phase of Raft Model Membranes from the Destructive Effect of Ionic Liquids.

Ionic liquids (ILs), although being a class of promising green solvents, have received many reports on the toxicity to living organisms. In this work, aiming at elucidating the disruptive effect of ILs to cell membrane lipid rafts, we investigated the effect of three 1-octylimidazolium-based ILs on the properties of the liquid ordered phase (Lo, a commonly used lipid raft model) of egg sphingomyelin (SM)-cholesterol model membrane. We found that, in the absence of cholesterol, a very low IL:SM molar ratio of 0.01:1 could disrupt the integrity of the bilayer structure. In sharp contrast, the presence of cholesterol in lipid bilayers helps the Lo phase resist the damaging effect of the ILs. For the role of the IL headgroup, we found that the mono- and trisubstituted species show a stronger destructive effect on the structures of the model rafts than the commonly used disubstituted counterpart.

Brillouin Spectroscopy of Binary Phospholipid-Cholesterol Bilayers.

Multicomponent lipid bilayers are used as models for searching the origin of spatial heterogeneities in biomembranes called lipid rafts, implying the coexistence of domains of different phases and compositions within the lipid bilayer. The spatial organization of multicomponent lipid bilayers on a scale of a hundred nanometers remains unknown. Brillouin spectroscopy providing information about the acoustic phonons with the wavelength of several hundred nanometers has an unexplored potential for this problem. Here, we applied Brillouin spectroscopy for three binary bilayers composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-palmitoyl-sn-glycero-3-phosphocholine (DPPC), and cholesterol. The Brillouin experiment for the oriented planar multibilayers was realized for two scattering geometries involving phonons for the lateral and normal directions of the propagation. The DPPC-DOPC mixtures known for the coexistence of the solid-ordered and liquid-disordered phases had bimodal Brillouin peaks, revealing the phase domains with sizes more than a hundred nanometers. Analysis of the Brillouin data for the binary mixtures concluded that the lateral phonons are preferable for testing the lateral homogeneity of the bilayers, while the phonons spreading across the bilayers are sensitive to the layered packing at the mesoscopic scale.