Fluorescence Studies of the Existence and Functional Importance of Regular Distributions in Liposomal Membranes

Abstract

Liposomes containing rigid and bulky lipid molecules such as sterols and pyrene‐labeled lipids can exhibit biphasic changes in membrane properties at several specific mole fractions predicted by the theory of lipid regular distributions (e.g., superlattices) in the plane of the membrane. This phenomenon has been observed in two‐component as well as multicomponent liquid‐crystalline liposomal membranes. The extent of sterol regular distribution plays a role in drug partitioning into membranes, the activities of surface‐acting enzymes such as cholesterol oxidase and phospholipase A2, and in free‐radical‐induced sterol oxidation. This article summarizes the original fluorescence studies of lipid superlattices, reviews the recent findings in this area, and discusses the current controversial issues related to lipid regular distributions.     

Critical factors for detection of biphasic changes in membrane properties at specific sterol mole fractions for maximal superlattice formation

Here we use the excitation generalized polarization (GPex) of 6-lauroyl-2-(dimethylamino)naphthalene (Laurdan) fluorescence in fluid cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine large unilamellar vesicles to explore the experimental conditions that would be required in order to detect a biphasic change in membrane properties at specific sterol mole fractions (Cr) (e.g., 20.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol %) for maximal sterol superlattice formation. Laurdan's GPex changes with sterol content in an alternating manner, showing minima (termed as GPex dips) at approximately Cr. GPex dips are detectable if the vesicles are preincubated for a sufficient time period and protected from sterol oxidation. In most cases, vesicles with a higher lipid concentration take a longer time to show a GPex dip at Cr. The depth of the GPex dip increases with increasing incubation time and eventually reaches a plateau, at which the maximum area covered by superlattices is expected to be achieved. However, if the vesicles are not protected against sterol oxidation, the GPex dips are attenuated or obliterated. These effects can be attributed to the increased inter-bilayer lipid exchange and the increased vesicle-vesicle interactions present at high lipid (vesicle) concentrations as well as the decreased interactions between oxysterols and phospholipids. These possible explanations have been incorporated into a kinetic model that is able to calculate the effects of sterol oxidation and lipid concentration on the depth of the GPex dip. The depth of the GPex dip, the required incubation time for the dip formation, and the lipid concentration dependence of the GPex dip vary with Cr, suggesting different physical properties for different sterol superlattices. To detect a biphasic change in membrane properties at Cr, one should also use small sterol mole fraction increments over a wide range, keep all of the vesicles in the same sample set under the same thermal history, and consider lipid concentration, probe type, and Cr value. These results improve our mechanistic understanding of sterol superlattice formation and explain why some studies, especially those requiring high lipid concentrations, did not detect a biphasic change in membrane properties at Cr.     

Cholesterol supports headgroup superlattice domain formation in fluid phospholipid/cholesterol bilayers

Fluorescence and Fourier transform infrared (FTIR) spectroscopic techniques were used to explore the effect of added cholesterol on the composition-dependent formation of putative phospholipid headgroup superlattices in fluid 1-palmitoyl-2-oleoyl-phosphatidylethanolamine/1-palmitoyl-2-oleoyl-phosphatidylcholine/cholesterol (POPE/POPC/CHOL) bilayers. Steady-state fluorescence anisotropy measurements of diphenylhexatriene (DPH) chain-labeled phosphatidylcholine (DPH-PC) revealed significant dips at several POPE-to-phospholipid mole fractions (X(PE)'s) when the cholesterol-to-lipid mole fraction (X(CHOL)) was fixed at 0.00, 0.35, 0.40, and 0.50. Most of the observed dips occur at or close to critical X(PE)'s predicted by the Headgroup Superlattice (SL) model, suggesting that phospholipid headgroups of different structures tend to adopt regular distributions even in the presence of cholesterol. Time-resolved fluorescence anisotropy measurements revealed that DPH-PC senses a disordered and highly mobile microenvironment in the POPE/POPC/CHOL bilayers at those critical X(PE)'s, indicating that this probe may partition to defect regions in the bilayers. The presence of coexisting packing defect regions and regularly distributed SL domains is a key feature predicted by the Headgroup SL model. Importantly, probe-free FTIR measurements of acyl chain C-H, interfacial carbonyl, and headgroup phosphate stretching peak frequencies revealed the presence of abrupt changes at X(PE)'s close to those observed in the fluorescence data. When X(PE) was varied from 0.60 to 0.72 and X(CHOL) from 0.34 to 0.46, a clear dip at the lipid composition coordinates (X(PE), X(CHOL)) approximately (0.68, 0.40) was observed in the three-dimensional surface plots of DPH-PC anisotropy as well as the carbonyl and phosphate stretching frequencies. The critical X(CHOL) at 0.40 agrees with the Cholesterol SL model, which assumes that cholesterol and phospholipid form SL domains at the lipid acyl chain level. In conclusion, this study provides evidence that cholesterol supports formation of phospholipid headgroup SLs in fluid state ternary lipid bilayers. The feasibility of the parallel existence of SLs at the lipid headgroup and acyl chain levels supports the relevance of the lipid SL model for the membranes of eukaryotic cells that typically contain significant amounts of cholesterol. We speculate that lipid SL formation may play a central role in the regulation of membrane lipid compositions, maintenance of organelle boundaries, and other crucial phenomena in those cells.     

Self-assembly and lipid interactions of diacylglycerol lactone derivatives studied at the air/water interface

Synthetic diacylglycerol lactones (DAG-lactones) have been shown to be effective modulators of critical cellular signaling pathways. The biological activity of these amphiphilic molecules depends in part upon their lipid interactions within the cellular plasma membrane. This study explores the thermodynamic and structural features of DAG-lactone derivatives and their lipid interactions at the air/water interface. Surface-pressure/area isotherms and Brewster angle microscopy revealed the significance of specific side-groups attached to the terminus of a very rigid 4-(2-phenylethynyl)benzoyl chain of the DAG-lactones, which affected both the self-assembly of the molecules and their interactions with phospholipids. The experimental data highlight the formation of different phases within mixed DAG-lactone/phospholipid monolayers and underscore the relationship between the two components in binary mixtures of different mole ratios. Importantly, the results suggest that DAG-lactones are predominantly incorporated within fluid phospholipid phases rather than in the condensed phases that form, for example, by cholesterol. Moreover, the size and charge of the phospholipid headgroups do not seem to affect DAG-lactone interactions with lipids.     

N-Myristoylated Phosphatidylethanolamine: Interfacial Behavior and Interaction with Cholesterol

The interfacial packing behavior of N-myristoyldimyristoylphosphatidylethanolamine (N-14:0 DMPE) and its interaction with cholesterol were characterized and compared to the behavior of dimyristoylphosphatidylethanolamine (DMPE) using an automated Langmuir type film balance. Surface pressure and surface potential were monitored as a function of lipid cross-sectional molecular area. N-14:0 DMPE exhibited two-dimensional (2D) phase transitions of a liquid-expanded to condensed nature at many temperatures in the 15-30 °C range, but isotherms showed only condensed behavior at 15 °C. The sharp decline in the surface compressional moduli upon entering the 2D-transition region is consistent with differences in the partial molar areas of coexisting liquid-expanded (chain-disordered) and condensed (chain-ordered) phases. Including Ca(2+) in the subphase beneath the negatively charged N-14:0 DMPE caused a downward shift in the 2D-transition onset pressure even in the presence of 100 mM NaCl. The average dipole moments perpendicular to the lipid-water interface for N-14:0 DMPE's liquid-expanded and condensed phases were higher than those of DMPE. At surface pressures sufficiently low (<10 mN/m) to produce liquid-expanded phase behavior in pure N-14:0 DMPE, mixing with cholesterol resulted in a classic "condensing effect". Maximal area condensation was observed near equimolar N-14:0 DMPE/cholesterol. Insights into mixing behavior at high surface pressures that mimic the lipid cross-sectional areas of biomembranes were provided by analyzing the surface compressional moduli as a function of cholesterol mole fraction. Complex mixing patterns were observed that deviated significantly from theoretical ideal mixing behavior suggesting the presence of lipid "complexes" and/or a liquid-ordered phase at high sterol mole fractions (>0.35) and low to intermediate surface pressures (<20 mN/m) as well as the possible coexistence of relatively immiscible solid phases at higher surface pressures (e.g., 35 mN/m).     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: EFFECTS OF CHOLESTEROL ON RADICAL YIELDS AND KINETIC PARAMETERS*

Abstract The quenching of the triplet state of chlorophyll a (Chl) by asymmetrically located electron acceptors was examined in vesicle systems containing egg yolk phosphatidylcholine and 0–50 mole % cholesterol. The incorporation of cholesterol had two main effects: (1) the distribution of Chl within the vesicle wall shifted from one favoring the inner monolayer to one favoring the outer monolayer, and (2) the Chi molecules (both ground and excited states) became more accessible to water and to the quencher molecules. This latter property was probably due to the creation of space between the phospholipid head groups by insertion of cholesterol. These phenomena required cholesterol concentrations in excess of 15 mol %. In general, the addition of cholesterol caused increases in the apparent bimolecular rate constant for triplet quenching, in the probability that quenching produced radicals, and in the rate of radical recombination. Some of the specific effects of cholesterol depended upon whether or not the quencher molecules were amphiphilic.     

Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation

The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity of the lipidated peptide to a fluid, DMPC-rich environment. This phase separation leads to the formation of peptide-containing domains with high fluorescence intensity that become progressively smaller with decreasing temperature. As a result, the local concentration of the peptide increases steadily within the confines of the shrinking domains. At the lowest temperatures, where the acyl-chain order parameter of the membrane has already drastically increased and the membrane achieves a liquid-ordered character, an efficient lipid sorting mechanism is no longer supported and aggregation of the peptide into small clusters prevails. We can conclude that palmitoyl/farnesyl dual-lipidated peptides do not associate with liquid-ordered or gel-like domains in phase-separated bilayer membranes. In particular, the study shows the interesting ability of the peptide to induce formation of fluid microdomains at physiologically relevant cholesterol concentrations, and this effect very much depends on the concentration of fluid vs ordered lipid molecules.     

Interaction of Polyunsaturated Fatty Acids with Cholesterol: A Role in Lipid Raft Phase Separation

Unequal affinity between lipids has been hypothesized to be a mechanism for the formation of microdomains/rafts in membranes. Our studies focus upon the interaction of cholesterol with polyunsaturated fatty acid (PUFA)-containing phospholipids. They support the proposal that steric incompatibility of the rigid steroid moiety for highly disordered PUFA chains, in particular docosahexaenoic acid (DHA), provides a sensitive trigger for lateral segregation of lipids into PUFA-rich/sterol-poor and PUFA-poor/sterol-rich regions. Solid state ²H NMR and x-ray diffraction (XRD) demonstrate that the solubility of cholesterol is reduced in 1-palmitoyl-2-docosahexaenoyl-phosphatidylethanolamine (16-0:22:6PE) bilayers. In mixed membranes of phosphatidylethanolamine (PE) with the lipid raft forming molecules egg sphingomyelin (SM) and cholesterol, diminished affinity of the sterol for 16:0-22:6PE relative to 1-palmitoyl-2-oleoylphosphatidylethanolamine (16:0-18:1PE) is identified by ²H NMR order parameters and detergent extraction. Phase separation of the PUFA-containing phospholipid from SM/cholesterol rafts is the implication, which may be associated with the myriad of health benefits of dietary DHA.     

Influence of organotin compounds on phosphatidylserine membranes

Organotin compounds are widely distributed toxicants. They are membrane‐active molecules with broad biological toxicity. We have studied the interaction of tributyltin and triphenyltin with phosphatidylserine model membranes using differential scanning calorimetry, infrared spectroscopy and X‐ray diffraction techniques. Organotin compounds produced a broadening of the gel to the liquid‐crystalline phase transition of the phospholipid and a shifting of the phase transition temperature to lower values. Infrared spectroscopy experiments showed that tributyltin exerted a fluidizing effect on the apolar part of the bilayer, and that both tributyl‐ and triphenyltin interact with the interfacial region of the bilayer, making the carbonyl groups less accessible to water. As seen by X‐ray diffraction experiments, organotin compounds were unable to change the bilayer macroscopic organization of the phospholipid, but they were able to reduce the long‐range order of the multibilayer system and to disorder the packing of the phospholipid molecules. The observed interaction between organotin compounds and phosphatidylserine membranes promotes physical perturbations that could affect membrane function and may mediate some of their toxic effects. Copyright © 2004 John Wiley & Sons, Ltd.     

Model of peripheral protein adsorption to the water/lipid interface

Two models have been developed to describe the adsorption of a model peripheral protein, colipase, to phospholipid/diacylglycerol (PL/DG) monolayers. One model is applicable at monolayer collapse pressure and at any composition that exceeds the DG mole fraction of PL/DG lateral complexes (Sugár, I. P.; Mizuno, N. K.; Momsen, M. M.; Brockman, H. L. Biophys. J. 2001, 81, 3387-3397). The other model is applicable at any lateral pressure but only below the mole fraction of DG in the complex (Sugár, I. P.; Mizuno, N. K.; Brockman, H. L. Biophys. J. 2005, 89, 3997-4005). Both models assume that initiation of colipase adsorption to the water/lipid interface requires an area of water-exposed hydrophobic surface that exceeds a critical value. In the first model, accessible surface is provided by the head groups of the uncomplexed DG molecules. This surface area follows a binomial distribution. In the second model, accessible area is created by hydrocarbon chains becoming exposed at the water/lipid interface as total lipid packing density of monolayers of PL and/or PL/DG complexes is decreased. This surface area follows a Poisson distribution. The model described in this paper is a unification, extension, and improvement of these models that is applicable at any lateral pressure and any PL/DG mole fraction. Calculated normalized initial colipase adsorption rates are compared with the available experimental values, and predictions of the adsorption rates are made for currently unmeasured compositions and lateral pressure regimes.     

Molecular dynamics studies of the molecular structure and interactions of cholesterol superlattices and random domains in an unsaturated phosphatidylcholine bilayer membrane

The effect of the molecular organization of lipid components on the properties of the bilayer membrane has been a topic of increasing interest. Several experimental and theoretical studies have suggested that cholesterol is not randomly distributed in the fluid-state lipid bilayer but forms nanoscale domains. Several cholesterol-enriched nanodomain structures have been proposed, including rafts, regular or maze arrays, complexes, and superlattices. At present, the molecular mechanisms by which lipid composition influences the formation and stability of lipid nanodomains remain unclear. In this study, we have used molecular dynamics (MD) simulations to investigate the effects of the molecular organization of cholesterol--superlattice versus random--on the structure of and interactions between lipids and water in lipid bilayers of cholesterol and 1-palmitoyl-2-oleoylphosphatidylcholine (cholesterol/POPC) at a fixed cholesterol mole fraction of 0.40. On the basis of four independent replicates of 200-ns MD simulations for a superlattice or random bilayer, statistically significant differences were observed in the lipid structural parameters, area per lipid, density profile, and acyl chain order profile, as well as the hydrogen bonding between various pairs (POPC and water, cholesterol and water, and POPC and cholesterol). The time evolution of the radial distribution of the cholesterol hydroxy oxygen suggests that the lateral distribution of cholesterol in the superlattice bilayer is more stable than that in the random bilayer. Furthermore, the results indicate that a relatively long simulation time, more than 100 ns, is required for these two-component bilayers to reach equilibrium and that this time is influenced by the initial lateral distribution of lipid components.     

Incorporation of blood clotting factor X into phospholipid model membranes: fluorescence microscopy imaging and subphase effects surrounding the lipid phase transition region

Factor X is a blood clotting protein that associates at membrane surfaces to become activated during the coagulation cascade. A molecular level understanding of the protein-membrane phospholipid interactions has not been reached, although it is thought that the protein binds to phospholipids in the presence of calcium through a bridge with the Gla (gamma-carboxyglutamic acid) domain on the protein. In this work, phospholipid Langmuir monolayers have been utilized as model membranes to study factor X association with phospholipid membrane components. Surface pressure measurements indicate that subphase addition of sodium, magnesium, and calcium ions enhances protein penetration of the lipid monolayer, with the largest association found with calcium ions in the subphase. Fluorescence microscopy images collected after protein penetration of lipid monolayers indicate monolayer condensation in the presence of sodium and magnesium ions. Aggregation of lipid domains is induced when calcium is in the subphase, indicating binding-induced flocculation of surface lipid aggregates. Calcium binding to factor X likely causes a conformational change which allows protein-membrane interaction via hydrophobic association with lipid molecules.     

Template-assisted self-assembly: a practical route to complex aggregates of monodispersed colloids with well-defined sizes, shapes, and structures

This paper describes a strategy that combines physical templating and capillary forces to assemble monodispersed spherical colloids into uniform aggregates with well-controlled sizes, shapes, and structures. When an aqueous dispersion of colloidal particles was allowed to dewet from a solid surface that had been patterned with appropriate relief structures, the particles were trapped by the recessed regions and assembled into aggregates whose structures were determined by the geometric confinement provided by the templates. We have demonstrated the capability and feasibility of this approach by assembling polystyrene beads and silica colloids (> or =150 nm in diameter) into complex aggregates that include polygonal or polyhedral clusters, linear or zigzag chains, and circular rings. We have also been able to generate hybrid aggregates in the shape of HF or H2O molecules that are composed of polymer beads having different diameters, polymer beads labeled with different organic dyes, and a combination of polymeric and inorganic beads. These colloidal aggregates can serve as a useful model system to investigate the hydrodynamic and optical scattering properties of colloidal particles having nonspherical morphologies. They should also find use as the building blocks to generate hierarchically self-assembled systems that may exhibit interesting properties highly valuable to areas ranging from photonics to condensed matter physics.     

Sterols associated with small unilamellar vesicles (SUVs): intrinsic mobility role for 1H NMR detection

Small unilamellar vesicles (SUVs) of phospholipids are often used as a membrane model system for studying the interaction of molecules. When using NMR under the standard liquid‐state conditions, SUV phospholipid proton spectra can be recorded, exhibiting sharp signals. This is not only because of the fast vesicular tumbling but also because of the combination of this tumbling with the individual motion of the lipids inside the bilayer. This appears evident because addition of cholesterol is responsible of broader resonances because of the slowing down of the lipid motion. On the other hand, no ¹H signal is detected for cholesterol in the bilayer. This lack of detection of the inserted molecules explains why generally SUVs are not considered as a good model for NMR studies under the standard liquid‐state conditions. Here, we use two other sterols in order to demonstrate that an increase of the molecular mobility inside the bilayer could allow the detection of their proton resonances. For desmosterol and lanosterol, which show higher mobility inside the bilayer, with increasing lateral diffusion rates, ¹H sterol signals are detected in contrast to cholesterol. For the fast diffusing lanosterol, no significant improvement in detection is observed using deuterated lipids, demonstrating that homonuclear dipolar coupling is fully averaged out. Furthermore, in the case of low mobility such as for cholesterol, the use of a fast magic angle spinning probe is shown to be efficient to recover the full proton spectrum. Copyright © 2014 John Wiley & Sons, Ltd.     

Aggregation and layering transitions in thin films of X-, T-, and anchor-shaped bolaamphiphiles at the air-water interface

Aggregation in Langmuir films is usually understood as being a disorderly grouping of molecules turning into chaotic three-dimensional aggregates and is considered an unwanted phenomenon causing irreversible changes. In this work we present the studies of 11 compounds from the group of specific surfactants, known as bolaamphiphiles, that exhibit reversible aggregation and, in many cases, transition to well-defined multilayers, which can be considered as a layering transition. These bolaamphiphiles incorporate rigid π-conjugated aromatics as hydrophobic cores, glycerol-based polar groups and hydrophobic lateral chains. Molecules of different shapes (X-, T-, and anchor) were studied and compared. The key property of these compounds is the partial fluorination of the lateral chains linked to the rigid cores of the molecules. The most interesting feature of the compounds is that, depending on their shape and degree of fluorination, they are able to resist aggregation and preserve a monolayer structure up to relatively high surface pressures (T-shaped and some X-shaped molecules), or create well-defined trilayers (X- and anchor-shaped molecules). Experimental studies were performed using Langmuir balance, surface potential and X-ray reflectivity measurements.     

N-(1-piperidinepropionyl)amphotericin B methyl ester (PAME)--a new derivative of the antifungal antibiotic amphotericin B: searching for the mechanism of its reduced toxicity

N-(1-piperidinepropionyl)amphotericin B methyl ester (in short, PAME), a low-toxicity amphotericin B derivative, has been investigated in Langmuir monolayers at the air/water interface alone and in mixtures with cellular membrane sterols (a mammalian sterol, cholesterol, and a fungal sterol, ergosterol) and a model phospholipid (DPPC). The analysis of the strength of interaction between PAME and both sterols as well as DPPC was based, on surface pressure measurements and analysis of the isothermal compressibility (C(s)(-1)), the mean area per molecule (A(12)), the excess free energy of mixing (DeltaG(Exc)) and the total free energy of mixing (DeltaG(M)). It has been found that the interactions between PAME and sterols are attractive; however, their strength is significantly weaker for mixtures of PAME with cholesterol than with ergosterol. This casts light on the improved selectivity of PAME toward fungal cells. The strongest interactions, found for PAME/DPPC mixtures, proved an important role of DPPC in the mechanism of reduced toxicity of PAME as compared to amphotericin B. Due to stable complex formation between PAME and DPPC the antibiotic is immobilized with DPPC molecules, which reduces the concentration of free antibiotic, which is capable of interacting with membrane sterols.     

Spontaneous vesicle formation of single chain and double chain cationic surfactant mixtures

The concentration vs composition diagram of aggregate formation of the dodecyltrimethylammonium bromide (DTAB) and didodecyldimethylammonium bromide (DDAB) mixture in aqueous solution at rather dilute region was constructed by analyzing the surface tension, turbidity, and electrical conductivity data and inspected by cryo-TEM images and dynamic light scattering data. Although the aqueous solution of DTAB forms only micelles, the transition from monomer to small aggregates and then to vesicle was found at 0.1 < X2 相似文献   

Replacing the cholesterol hydroxyl group with the ketone group facilitates sterol flip-flop and promotes membrane fluidity

The 3alpha-hydroxyl group is a characteristic structural element of all membrane sterol molecules, while the 3-ketone group is more typically found in steroid hormones. In this work, we investigate the effect of substituting the hydroxyl group in cholesterol with the ketone group to produce ketosterone. Extensive atomistic molecular dynamics simulations of saturated lipid membranes with either cholesterol or ketosterone show that, like cholesterol, ketosterone increases membrane order and induces condensation. However, the effect of ketosterone on membrane properties is considerably weaker than that of cholesterol. This is largely due to the unstable positioning of ketosterone at the membrane-water interface, which gives rise to a small but significant number of flip-flop transitions, where ketosterone is exchanged between membrane leaflets. This is remarkable, as flip-flop motions of sterol molecules have not been previously reported in analogous lipid bilayer simulations. In the same context, ketosterone is found to be more tilted with respect to the membrane normal than cholesterol. The atomic level mechanism responsible for the increase of the steroid tilt and the promotion of flip-flops is the decrease in polar interactions at the membrane-water interface. Interactions between lipids or water and the ketone group are found to be weaker than in the case of the hydroxyl group, which allows ketosterone to penetrate through the hydrocarbon region of a membrane.     

Lateral diffusion of molecules in two-component lipid bilayer: a Monte Carlo simulation study

Lateral diffusion of membrane components makes possible any in-plane membrane reaction and has a key role in signaling in cell membranes. In this report the equilibrium lateral diffusion of intrinsic molecules in an equimolar DMPC/DSPC mixture is simulated using a thoroughly tested two-state model of two-component phospholipid bilayers. The model has been successful in calculating the excess heat capacity function, the most frequent center-to-center distances between DSPC clusters, and the fractal dimensions of gel clusters (Sugar, I. P., Thompson, T. E., Biltonen, R. L. Biophys. J. 1999, 76, 2099-2110). In the gel/fluid mixed phase region, a diffusing intrinsic molecule may change its state from fluid to gel (or from gel to fluid) at any time. A common characterization of the diffusion of intrinsic molecules is given by the simulated average first-passage time curves. We find that these curves can be described as power functions containing two parameters, alpha and beta, except near the percolation threshold of gel/fluid or compositional clusters. We find also that the intrinsic molecules are involved in approximately normal diffusion, i.e., beta approximately 2 in the extreme gel and fluid phase regions, while in the gel/fluid and gel/gel mixed phase regions the diffusion is anomalous, i.e., beta not equal 2. In the mixed phase regions, when the initial local state of the diffusing molecule is not specified, each component is involved in sub-diffusion (beta > 2). In the gel/fluid mixed phase region molecules situated initially inside a fluid cluster are involved in sub-diffusion, but DMPC molecules situated initially inside a gel cluster are involved in super-diffusion (beta < 2). The possibility of anomalous diffusion in membranes apparently arises because the diffusing molecule visits a variety of different environments characterized by its relative proximity to various membrane components. The diffusion is actually anomalous when the components of the bilayer are nonrandomly distributed. The deviation from random distribution is strongly correlated with beta. Similar to the results of the NMR experiments, the calculated relative diffusion coefficient continuously decreases in the gel/fluid mixed phase region with decreasing temperature. In apparent contradiction, diffusion measured by fluorescence recovery after photobleaching (FRAP) demonstrates the existence of a threshold temperature, below which long-range diffusion of FRAP probe molecules is essentially blocked. This threshold temperature is highly correlated with the percolation temperature of gel clusters.     

High-affinity small molecule-phospholipid complex formation: binding of siramesine to phosphatidic acid

Siramesine (SRM) is a sigma-2 receptor agonist which has been recently shown to inhibit growth of cancer cells. Fluorescence spectroscopy experiments revealed two distinct binding sites for this drug in phospholipid membranes. More specifically, acidic phospholipids retain siramesine on the bilayer surface due to a high-affinity interaction, reaching saturation at an apparent 1:1 drug-acidic phospholipid stoichiometry, where after the drug penetrates into the hydrocarbon core of the membrane. This behavior was confirmed using Langmuir films. Of the anionic phospholipids, the highest affinity, comparable to the affinities for the binding of small molecule ligands to proteins, was measured for phosphatidic acid (PA, mole fraction of X(PA) = 0.2 in phosphatidylcholine vesicles), yielding a molecular partition coefficient of 240 +/- 80 x 10(6). An MD simulation on the siramesine:PA interaction was in agreement with the above data. Taking into account the key role of PA as a signaling molecule promoting cell growth our results suggest a new paradigm for the development of anticancer drugs, viz. design of small molecules specifically scavenging phospholipids involved in the signaling cascades controlling cell behavior.