Nanoscale electrostatic domains in cholesterol-laden lipid membranes create a target for amyloid binding

Amyloid fibrils are associated with multiple neurodegenerative disorders, such as Alzheimer's disease. Although biological membranes are involved in fibril plaque formation, the role of lipid membrane composition in fibril formation and toxicity is not well understood. We investigated the effect of cholesterol on the interaction of model lipid membranes with amyloid-β peptide (Aβ). With atomic force microscopy we demonstrated that binding of Aβ (1-42) to DOPC bilayer, enriched with 20% cholesterol, resulted in an intriguing formation of small nonuniform islands loaded with Aβ. We attribute this effect to the presence of nanoscale electrostatic domains induced by cholesterol in DOPC bilayers. Using frequency-modulated Kelvin probe force microscopy we were able to resolve these nanoscale electrostatic domains in DOPC monolayers. These findings directly affect our understanding of how the presence of cholesterol may induce targeted binding of amyloid deposits to biomembranes. We postulate that this nonhomogeneous electrostatic effect of cholesterol has a fundamental nature and may be present in other lipid membranes and monolayers.     

The effects of various membrane physical-chemical properties on the aggregation kinetics of insulin

In a simplified approach to the in vivo situation, where pathogenic fibrillar protein deposits are often found associated with cellular membranes, the aggregation kinetics of insulin in the presence of various model biomembranes were investigated using the Thioflavin T (ThT) fluorescence assay. The lipid dynamics near the gel-fluid transition, the chain length of saturated lipids and the presence of DOPE or DOPS in DOPC-vesicles modulate the aggregation kinetics of insulin in an indifferent, an aggregation-accelerating or an aggregation-inhibiting manner, subtly depending on the pH-value and the presence of salt. The rate of insulin aggregation in bulk solution dominates the overall aggregation process in most cases at low pH, where the lipid additives exert no effect on the aggregation kinetics. The occurrence of dynamic line defects near the gel-fluid transition temperature of DSPC facilitates a partial membrane insertion of the protein, which in turn shields exposed hydrophobic protein patches from intermolecular association and hence inhibit aggregation. An exclusively aggregation-accelerating effect was observed in the presence of 0.1M NaCl for all lipid additives investigated, which is likely due to an enhanced surface accumulation of the protein. Apart from weak dipole-dipole, dipole-monopole and hydrogen bonding interactions, the release of curvature elastic stress in mixed DOPC/DOPE-membranes and preferred interactions of insulin with carboxylic groups in DOPC/DOPS-membranes favour an increased surface accumulation. At neutral pH, a partial insertion of insulin into the lipid bilayer is favoured, which accounts for the aggregation-inhibiting effect of all lipid bilayer systems studied except those containing DOPS. Generally, the extent of inhibition increases with the lipid chain length and the extent of curvature stress in mixed unsaturated lipid membranes and also when the gel-fluid transition temperature of pure phospholipids is approached. The accelerating effect of DOPS on the aggregation of insulin under net electrostatic repulsion at pH 7.4 remains to be elucidated, yet, it might result from increased surface accumulation and/or faster/more extensive unfolding of the protein without a subsequent membrane insertion. These results demonstrate that a delicate interplay between different physical and chemical properties of lipid membranes has to be taken into account in a detailed discussion of membrane-associated protein aggregation phenomena.     

The Nonsteroidal Anti-Inflammatory Drug Indomethacin Induces Heterogeneity in Lipid Membranes: Potential Implication for Its Diverse Biological Action

Background

The nonsteroidal anti-inflammatory drug (NSAID), indomethacin (Indo), has a large number of divergent biological effects, the molecular mechanism(s) for which have yet to be fully elucidated. Interestingly, Indo is highly amphiphilic and associates strongly with lipid membranes, which influence localization, structure and function of membrane-associating proteins and actively regulate cell signaling events. Thus, it is possible that Indo regulates diverse cell functions by altering micro-environments within the membrane. Here we explored the effect of Indo on the nature of the segregated domains in a mixed model membrane composed of dipalmitoyl phosphatidyl-choline (di16∶0 PC, or DPPC) and dioleoyl phosphatidyl-choline (di18∶1 PC or DOPC) and cholesterol that mimics biomembranes.

Methodology/Principal Findings

Using a series of fluorescent probes in a fluorescence resonance energy transfer (FRET) study, we found that Indo induced separation between gel domains and fluid domains in the mixed model membrane, possibly by enhancing the formation of gel-phase domains. This effect originated from the ability of Indo to specifically target the ordered domains in the mixed membrane. These findings were further confirmed by measuring the ability of Indo to affect the fluidity-dependent fluorescence quenching and the level of detergent resistance of membranes.

Conclusion/Significance

Because the tested lipids are the main lipid constituents in cell membranes, the observed formation of gel phase domains induced by Indo potentially occurs in biomembranes. This marked Indo-induced change in phase behavior potentially alters membrane protein functions, which contribute to the wide variety of biological activities of Indo and other NSAIDs.     

Binding of peripheral proteins to mixed lipid membranes: effect of lipid demixing upon binding

Binding isotherms have been determined for the association of horse heart cytochrome c with dioleoyl phosphatidylglycerol (DOPG)/dioleoyl phosphatidylcholine (DOPC) bilayer membranes over a range of lipid compositions and ionic strengths. In the absence of protein, the DOPG and DOPC lipids mix nearly ideally. The binding isotherms have been analyzed using double layer theory to account for the electrostatics, either the Van der Waals or scaled particle theory equation of state to describe the protein surface distribution, and a statistical thermodynamic formulation consistent with the mass-action law to describe the lipid distribution. Basic parameters governing the electrostatics and intrinsic binding are established from the binding to membranes composed of anionic lipid (DOPG) alone. Both the Van der Waals and scaled particle equations of state can describe the effects of protein distribution on the DOPG binding isotherms equally well, but with different values of the maximum binding stoichiometry (13 lipids/protein for Van der Waals and 8 lipids/protein for scaled particle theory). With these parameters set, it is then possible to derive the association constant, Kr, of DOPG relative to DOPC for surface association with bound cytochrome c by using the binding isotherms obtained with the mixed lipid membranes. A value of Kr (DOPG:DOPC) = 3.3-4.8, depending on the lipid stoichiometry, is determined that consistently describes the binding at different lipid compositions and different ionic strengths. Using the value of Kr obtained it is possible to derive the average in-plane lipid distribution and the enhancement in protein binding induced by lipid redistribution using the statistical thermodynamic theory.     

Interaction of the NMDA receptor noncompetitive antagonist MK-801 with model and native membranes.

MK-801, a noncompetitive antagonist of the NMDA (N-methyl-D-aspartate) receptor, has protective effects against excitotoxicity and ethanol withdrawal seizures. We have determined membrane/buffer partition coefficients (Kp[mem]) of MK-801 and its rates of association with and dissociation from membranes. Kp[mem] (+/- SD) = 1137 (+/- 320) in DOPC membranes and 485 (+/- 99) in synaptoneurosomal (SNM) lipid membranes from rat cerebral cortex (unilamellar vesicles). In multilamellar vesicles, Kp[mem] was higher: 3374 (+/- 253) in DOPC and 6879 (+/- 947) in SNM. In cholesterol/DOPC membranes, Kp[mem] decreased as the cholesterol content increased. MK-801 associated with and dissociated from membranes rapidly. Addition of ethanol to SNM did not affect Kp[mem]. MK-801 decreased the cooperative unit size of DMPC membranes. The decrease was smaller than that caused by 1,4-dihydropyridine drugs, indicating a weaker interaction with the hydrocarbon core. Small angle x-ray diffraction, with multilayer autocorrelation difference function modeling, indicated that MK-801 in a cholesterol/DOPC membrane (mole ratio = 0.6) causes a perturbation at approximately 16.0 A from the bilayer center. In bilayers of cholesterol/DOPC = 0.15 (mole ratio) or pure DOPC, the perturbation caused by MK-801 was more complex. The physical chemical interactions of MK-801 with membranes in vitro are consistent with a fast onset and short duration of action in vivo.     

Properties of Hexadecaprenyl Monophosphate/Dioleoylphosphatidylcholine Vesicular Lipid Bilayers

In our study we investigated hemispherical phospholipid bilayer membranes and phospholipid vesicles made from hexadecaprenyl monophosphate (C₈₀-P), dioleoylphosphatidylocholine (DOPC) and their mixtures by voltammetric and transmission electron microscopy (TEM) techniques. The current-voltage characteristics, the membrane conductance-temperature relationships and the membrane breakdown voltage have been measured for different mixtures of C₈₀-P/DOPC. The membrane hydrophobic thickness and the activation energy of ion migration across the membrane have been determined. Hexadecaprenyl monophosphate decreased in comparison with DOPC bilayers, the membrane conductance, increased the activation energy and the membrane breakdown voltage for the various value of C₈₀-P/DOPC mole ratio, respectively. The TEM micrographs of C₈₀-P, DOPC and C₈₀-P/DOPC lipid vesicles showed several characteristic structures, which have been described. The data indicate that hexadecaprenyl monophosphate modulates the surface curvature of the membranes by the formation of aggregates in liquid-crystalline phospholipid membranes. We suggest that the dynamics and conformation of hexadecaprenyl monophosphate in membranes depend on the transmembrane electrical potential. The electron micrographs indicate that polyprenyl monophosphates with single isoprenyl chains form lipid vesicular bilayers. The thickness of the bilayer, evaluated from the micrographs, was 11 ± 1 nm. This property creates possibility of forming primitive bilayer lipid membranes by long single-chain polyprenyl phosphates in abiotic conditions. It can be the next step in understanding the origin of protocells. Received: 10 January 2000/Revised: 7 June 2000     

Influence of lipid composition on physical properties and peg-mediated fusion of curved and uncurved model membrane vesicles: "nature's own" fusogenic lipid bilayer

Poly(ethylene glycol) (PEG)-mediated fusion of phosphatidylcholine model membranes has been shown to mimic the protein-mediated biomembrane process [Lee, J., and Lentz, B. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9274-9279]. Unlike the simple model membranes used in this earlier study, the lipid composition of fusogenic biomembranes is quite complex. The purpose of this paper was to examine PEG-mediated fusion of highly curved (SUV) and largely uncurved (LUV) membrane vesicles composed of different lipids in order to identify lipid compositions that produce highly fusogenic membranes. Starting with liposomes composed of five lipids with different physical properties, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE), dioleoylphosphatidylserine (DOPS), bovine brain sphingomyelin (SM), and cholesterol (CH), we systematically varied the composition and tested for the extent of PEG-mediated fusion after 5 min of treatment. We found that a vesicle system composed of four lipids, DOPC/DOPE/SM/CH, fused optimally at a 35/30/15/20 molar ratio. Each lipid seemed to play a part in optimizing the membrane for fusion. PE disrupted outer leaflet packing as demonstrated with TMA-DPH lifetime, C(6)-NBD-PC partitioning, and DPH anisotropy measurements, and thus significantly enhanced fusion and rupture, without significantly altering interbilayer approach (X-ray diffraction). An optimal ratio of PC/PE (35/30) produced a balance between fusion and rupture. CH and SM, when present at an optimal ratio of 3/4 in vesicles containing the optimal PC/PE ratio, reduced rupture without significantly reducing fusion. This optimal CH/SM ratio also enhanced outer leaflet packing, suggesting that fusion is dependent not only on outer leaflet packing but also on the properties of the inner leaflet. Addition of CH without SM enhanced rupture relative to fusion, while SM alone reduced both rupture and fusion. The optimal lipid composition is very close to the natural synaptic vesicle composition, suggesting that the synaptic vesicle composition is optimized with respect to fusogenicity.     

Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.     

Morphological and Physical Analysis of Natural Phospholipids-Based Biomembranes

Background

Liposomes are currently an important part of biological, pharmaceutical, medical and nutritional research, as they are considered to be among the most effective carriers for the introduction of various types of bioactive agents into target cells.

Scope of Review

In this work, we study the lipid organization and mechanical properties of biomembranes made of marine and plant phospholipids. Membranes based on phospholipids extracted from rapeseed and salmon are studied in the form of liposome and as supported lipid bilayer. Dioleylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine (DPPC) are used as references to determine the lipid organization of marine and plant phospholipid based membranes. Atomic force microscopy (AFM) imaging and force spectroscopy measurements are performed to investigate the membranes'' topography at the micrometer scale and to determine their mechanical properties.

Major Conclusions

The mechanical properties of the membranes are correlated to the fatty acid composition, the morphology, the electrophoretic mobility and the membrane fluidity. Thus, soft and homogeneous mechanical properties are evidenced for salmon phospholipids membrane containing various polyunsaturated fatty acids. Besides, phase segregation in rapeseed membrane and more important mechanical properties were emphasized for this type of membranes by contrast to the marine phospholipids based membranes.

General Significance

This paper provides new information on the nanomechanical and morphological properties of membrane in form of liposome by AFM. The originality of this work is to characterize the physico-chemical properties of the nanoliposome from the natural sources containing various fatty acids and polar head.     

From small fluctuations to large-scale phase separation: lateral organization in model membranes containing cholesterol

Macroscopic coexisting liquid phases are readily observed in certain model membranes containing cholesterol and at least two other lipid components. Recent fluorescence microscopy and deuterium NMR work indicates that submicron fluctuations are also found, in vesicles with near-critical lipid compositions. In principle, the magnitude of critical fluctuations can be controlled by changing temperature, or through other means of shifting the phase boundary such as including impurities or cross-linking components. Critical fluctuations are dynamic submicron domains in model membranes, and provide a plausible physical mechanism to produce putative 'raft' domains in cholesterol-rich biomembranes.     

Effects of unsaturation and curvature on the transverse distribution of intramolecular dynamics of dipyrenyl lipids.

The roles of acyl chain unsaturation and curvature in the excimer formation efficiency (EFE) of site-specific conjugated pyrene molecules in lipid membranes have been investigated by steady-state and time-resolved fluorescence spectroscopy. Six 1-2-(pyrenyl-n-acyl)-phosphatidylcholine (dipy(n)PC) probes, with pyrenyl chains of varying methylene units n from 4 to 14 carbons, were incorporated separately into dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylethanolamine (DOPE) lipid membranes at 0.1 mol%. Both the excimer-to-monomer fluorescence intensity ratio and association-to-dissociation rate constant ratio of conjugated pyrenes were used to quantify EFE. At all temperatures (T = 0-30 degrees C) and for n = 4 and 6, the EFE for DOPE was always smaller than EFE for DOPC. At T < 10 degrees C (where DOPE and DOPC are in the liquid crystalline L alpha phase) and for n > 8, the EFE for curvature frustrated DOPE was significantly greater than EFE for nonfrustrated DOPC (control), and the difference increased gradually with n. At T> 18 degrees C (where DOPE is in the inverted hexagonal H(II) phase and DOPC is in the L alpha phase) and for n > 8, EFE for the curvature-relaxed DOPE was again smaller than the EFE for DOPC control. The contributions of splay conformation and internal dynamics of pyrenyl chains to EFE were examined separately using a lattice model. Our results suggest that i) the cis double bonds of the host lipid matrix strongly perturb both the conformation and dynamics of conjugated pyrenes at the specific location around n = 8, and ii) the lateral stress at the upper part (n < 8) of the curvature frustrated bilayer membranes (DOPE) may be significantly relaxed once the membrane surface adopts a favorable negative interfacial curvature.     

The effect of long-chain bases on polysialic acid-mediated membrane interactions

Negatively-charged polysialic acid (polySia) chains are usually membrane-bound and are often expressed on the surface of neuroinvasive bacterial cells, neural cells, and tumor cells. PolySia can mediate both repulsive and attractive cis interactions between membrane components, and trans interactions between membranes. Positively-charged long-chain bases are widely present in cells, are often localized in membranes and can function as bioactive lipids. Here we use Langmuir monolayer technique, fluorescence spectroscopy and electron microscopy of lipid vesicles to study the role of a simple long-chain base, octadecylamine (ODA), in both cis and trans interactions mediated by polySia in model membranes composed of ODA and dioleoylphospatidycholine (DOPC). When added free to an aqueous solution, polySia increases the collapse pressure of ODA/DOPC monolayers, reduces the effect of ODA on the limiting molecular area, inverses the values of excess area per molecule and of excess free energy of mixing from positive to negative, and induces fusion of ODA/DOPC vesicles. These results suggest that a polySia chain can act as a multi-bridge that mediates cis interactions between different components of a lipid membrane, disrupts membrane aggregates, and mediates trans interactions between lipids in apposing membranes. These observations imply that polySia in cellular systems can act in a similar way.     

Lipid distribution and transport across cellular membranes

In eukaryotic cells, the membranes of different intracellular organelles have different lipid composition, and various biomembranes show an asymmetric distribution of lipid types across the membrane bilayer. Membrane lipid organization reflects a dynamic equilibrium of lipids moving across the bilayer in both directions. In this review, we summarize data supporting the role of specific membrane proteins in catalyzing transbilayer lipid movement, thereby controlling and regulating the distribution of lipids over the leaflets of biomembranes.     

Analysis of membrane interactions of antibiotic peptides using ITC and biosensor measurements

The interaction of the lantibiotic gallidermin and the glycopeptide antibiotic vancomycin with bacterial membranes was simulated using mass sensitive biosensors and isothermal titration calorimetry (ITC). Both peptides interfere with cell wall biosynthesis by targeting the cell wall precursor lipid II, but differ clearly in their antibiotic activity against individual bacterial strains. We determined the binding affinities of vancomycin and gallidermin to model membranes±lipid II in detail. Both peptides bind to DOPC/lipid II membranes with high affinity (K(D) 0.30 μM and 0.27 μM). Gallidermin displayed also strong affinity to pure DOPC membranes (0.53 μM) an effect that was supported by ITC measurements. A surface acoustic wave (SAW) sensor allowed measurements in the picomolar concentration range and revealed that gallidermin targets lipid II at an equimolar ratio and simultaneously inserts into the bilayer. These results indicate that gallidermin, in contrast to vancomycin, combines cell wall inhibition and interference with the bacterial membrane integrity for potent antimicrobial activity.     

Order parameters of unsaturated phospholipids in membranes and the effect of cholesterol: a 1H–13C solid-state NMR study at natural abundance

Most biological phospholipids contain at least one unsaturated alkyl chain. However, few order parameters of unsaturated lipids have been determined because of the difficulty associated with isotopic labeling of a double bond. Dipolar recoupling on axis with scaling and shape preservation (DROSS) is a solid-state nuclear magnetic resonance technique optimized for measuring ¹H–¹³C dipolar couplings and order parameters in lipid membranes in the fluid phase. It has been used to determine the order profile of 1,2-dimyristoyl-sn-glycero-3-phosphocholine hydrated membranes. Here, we show an application for the measurement of local order parameters in multilamellar vesicles containing unsaturated lipids. Taking advantage of the very good ¹³C chemical shift dispersion, one can easily follow the segmental order along the acyl chains and, particularly, around the double bonds where we have been able to determine the previously misassigned order parameters of each acyl chain of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We have followed the variation of such order profiles with temperature, unsaturation content and cholesterol addition. We have found that the phase formed by DOPC with 30% cholesterol is analogous to the liquid-ordered (l_o) phase. Because these experiments do not require isotopic enrichment, this technique can, in principle, be applied to natural lipids and biomembranes.Electronic Supplementary Material Supplementary material is available for this article at .     

Bulk properties of the lipid bilayer are not essential for the thermal stability of Na,K-ATPase from shark rectal gland or pig kidney

The thermal stability of Na,K-ATPase from pig kidney is markedly greater than that of Na,K-ATPase from shark salt glands. The role of the lipid bilayer is studied by solubilisation of the membrane-bound enzyme in the nonionic detergent octaethyleneglycoldodecylmonoether (C₁₂E₈), addition of excess dioleylphosphatidylcholine (DOPC) or palmitoyloleylphosphatidylcholine (POPC) and reconstitution of membranes by removal of detergent. At 54 °C the reconstituted enzymatically active pig enzyme retains a high thermal stability, and reconstituted shark enzyme retains a low thermal stability, even with a 9-fold excess of DOPC. This result suggests that the origin of the difference in thermal stability is not related to bulk lipid properties of the native membranes.     

Phospholipid flop induced by transmembrane peptides in model membranes is modulated by lipid composition

Since phospholipid synthesis is generally confined to one leaflet of a membrane, membrane growth requires phospholipid translocation (flip-flop). It is generally assumed that this process is protein-mediated; however, the mechanism of flip-flop remains elusive. Previously, we have demonstrated flop of 2-[6-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]caproyl] (C6NBD) phospholipids, induced by the presence of membrane-spanning peptides in vesicles composed of an Escherichia coli phospholipid extract, supporting the hypothesis that the presence of transmembrane stretches of proteins in the bilayer is sufficient to allow phospholipid flip-flop in the inner membrane of E. coli [Kol et al. (2001) Biochemistry 40, 10500]. Here, we investigated whether the specific phospholipid composition of E. coli is a prerequisite for transmembrane helix-induced flop of phospholipids. This was tested by determining the amount of C6NBD-phospholipid that was translocated from the inner leaflet to the outer leaflet of a model membrane in time, using a dithionite reduction assay. The transmembrane peptides GWWL(AL)8WWA (WALP23) and GKKL(AL)8KKA (KALP23) induced phospholipid flop in model membranes composed of various lipid mixtures. The rate of peptide-induced flop was found to decrease with increasing dioleoylphosphatidylethanolamine (DOPE) content of vesicles composed of DOPE and dioleoylphosphatidylcholine (DOPC), and the rate of KALP23-induced flop was shown to be stimulated by higher dioleoylphosphatidylglycerol (DOPG) content in model membranes composed of DOPG and DOPC. Furthermore, the incorporation of cholesterol had an inhibitory effect on peptide-induced flop. Finally, flop efficiency was strongly dependent on the phospholipid headgroup of the NBD-phospholipid analogue. Possible implications for transmembrane helix-induced flop in biomembranes in general are discussed.     

Acyl-chain methyl distributions of liquid-ordered and -disordered membranes

A central feature of the lipid raft concept is the formation of cholesterol-rich lipid domains. The introduction of relatively rigid cholesterol molecules into fluid liquid-disordered (L_d) phospholipid bilayers can produce liquid-ordered (L_o) mixtures in which the rigidity of cholesterol causes partial ordering of the flexible hydrocarbon acyl chains of the phospholipids. Several lines of evidence support this concept, but direct structural information about L_o membranes is lacking. Here we present the structure of L_o membranes formed from cholesterol and dioleoylphosphatidylcholine (DOPC). Specific deuteration of the DOPC acyl-chain methyl groups and neutron diffraction measurements reveal an extraordinary disorder of the acyl chains of neat L_d DOPC bilayers. The disorder is so great that >20% of the methyl groups are in intimate contact with water in the bilayer interface. The ordering of the DOPC acyl chains by cholesterol leads to retraction of the methyl groups away from the interface. Molecular dynamics simulations based on experimental systems reveal asymmetric transbilayer distributions of the methyl groups associated with each bilayer leaflet.     

Interactions of cholesterol hemisuccinate with phospholipids and (Ca2+-Mg2+)-ATPase

Cholesterol hemisuccinate has been shown to equilibrate readily with liposomes and with the (Ca2+-Mg2+)-ATPase from sarcoplasmic reticulum and has been used to modify the sterol content of these membranes. Cholesterol hemisuccinate incorporates into dioleoylphosphatidylcholine (DOPC) up to a molar ratio of 3:1 sterol to DOPC. Effects on lipid order as detected by electron spin resonance and fluorescence polarization are comparable to those of cholesterol. Binding constants have been determined, and the uncharged form of the sterol binds more strongly than the anionic form. Binding to DOPC and to the lipid component of the ATPase system is comparable. From use of the fluorescence quenching properties of 1,2-bis(9,10- dibromooleoyl )phosphatidylcholine and dibromocholesterol hemisuccinate, two classes of binding sites on the ATPase have been deduced. At the lipid/protein interface, the binding constant for cholesterol hemisuccinate is considerably less than that for DOPC. At the second set of sites ( nonannular sites), binding occurs with Kd = 0.55 in molar ratio units. The effect of cholesterol hemisuccinate on the activity of the ATPase depends on the phospholipid present in the system: ATPase reconstituted with DOPC is inhibited whereas ATPase reconstituted with dimyristoleoylphosphatidylcholine is activated. We conclude that changes in membrane fluidity are not important in determining ATPase activity in these systems.     

A new method for measuring edge tensions and stability of lipid bilayers: effect of membrane composition

We report a novel and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (eggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of DOPC with cholesterol and dioleoylphosphatidylethanolamine. Our data confirm previous results for eggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of dioleoylphosphatidylethanolamine is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. It is presumed that interlipid hydrogen bonding is the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner.