Acyl-coenzyme A organizes laterally in membranes and is recognized specifically by acyl-coenzyme A binding protein.

Long chain acyl-coenzyme A (acyl-CoA) is a biochemically important amphiphilic molecule that is known to partition strongly into membranes by insertion of the acyl chain. At present, microscopically resolved evidence is lacking on how acyl-CoA influences and organizes laterally in membranes. By atomic force microscopy (AFM) imaging of membranes exposed to acyl-CoA in microM concentrations, it is shown that aggregate formation takes place within the membrane upon long-time exposure. It is known that acyl-CoA is bound by acyl-CoA binding protein (ACBP) with high affinity and specificity and that ACBP may bind and desorb membrane-bound acyl-CoA via a partly unknown mechanism. Following incubation with acyl-CoA, it is shown that ACBP is able to reverse the formation of acyl-CoA aggregates and to associate peripherally with acyl-CoA on the membrane surface. Our microscopic results point to the role of ACBP as an intermembrane transporter of acyl-CoA and demonstrate the ability of AFM to reveal the remodelling of membranes by surfactants and proteins.

Association of acylated cationic decapeptides with dipalmitoylphosphatidylserine-dipalmitoylphosphatidylcholine lipid membranes.

The interaction of three acylated and cationic decapeptides with lipid membranes composed of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylserine (DPPS) has been studied by means of fluorescence spectroscopy and differential scanning calorimetry (DSC). The synthetic model decapeptides that are N-terminally linked with C(2), C(8), and C(14) acyl chains contain four basic histidine residues in their identical amino acid sequence. A binding model, based on changes in the intrinsic fluorescent properties of the peptides upon association with the DPPC-DPPS membranes, is used to estimate the peptide-membrane dissociation constants. The results clearly show that all three peptides have a higher affinity to liposomes containing DPPS lipids due to non-specific electrostatic interactions between the cationic peptides and the anionic DPPS lipids. Furthermore, it is found that the acyl chain length of the peptides plays a crucial role for the binding. A preference for fluid phase membranes as compared to gel phase membranes is generally observed for all three peptides. DSC is used to characterise the influence of the three peptides on the thermodynamic phase behaviour of the binary DPPC-DPPS lipid mixture. The extent of peptide association deduced from the heat capacity measurements suggests a strong binding and membrane insertion of the C(14) acylated peptide in accordance with the fluorescence measurements.

Phospholipase A(2) activity towards vesicles of DPPC and DMPC-DSPC containing small amounts of SMPC.

Phospholipase A(2) (PLA(2)) is an interfacially active enzyme whose hydrolytic activity is known to be enhanced in one-component phospholipid bilayer substrates exhibiting dynamic micro-heterogeneity. In this study the activity of PLA(2) towards large unilamellar vesicles composed of DPPC:SMPC and DMPC:DSPC:SMPC is investigated using fluorescence and HPLC techniques. Phase diagrams of the mixtures are established by differential scanning calorimetry and the PLA(2) activity, monitored by the lag time, is correlated with the phase behavior of the mixtures. In addition, the degree of lipid hydrolysis in the DMPC:DSPC:SMPC lipid mixtures is detected by HPLC. The PLA(2) activity is found to be significantly increased in the temperature range of the coexistence region where the lipid mixtures exhibit lateral gel-fluid phase separation. Furthermore, in the entire temperature range it is demonstrated that PLA(2) preferentially hydrolyzes the short chain DMPC lipid. This discriminative effect becomes less pronounced when the asymmetric lipid SMPC is present in the lipid substrate. Inclusion of SMPC into either DPPC or DMPC:DSPC vesicles prolongs the lag time. The results clearly show that the PLA(2) activity is significantly enhanced by lipid bilayer micro-heterogeneity in both one-component and multi-component lipid bilayer substrates. The PLA(2) activity measurements are discussed in terms of dynamic gel-fluid lipid domain formation due to density fluctuations and static lipid domain formation due to gel-fluid phase separation.

Dynamical and structural properties of lipid membranes in relation to liposomal drug delivery systems.

The structural and dynamical properties of DPPC liposomes containing lipopolymers (PEG-lipids) and charged DPPS lipids have been studied in relation to the lipid membrane interaction of enzymes and peptides. The results suggest that both the lipid membrane structure and dynamics and in particular the appearance of small-scale lipid structures might be of importance for the activity of membrane associated and liposome degrading enzymes as well as for the membrane interaction of acylated peptides. The combined experimental and simulation results are of relevance for a rational development of peptide loaded liposomal drug delivery systems that become destabilized by membrane degrading phospholipase A(2) enzymes, which are found at elevated concentrations at diseased sites.

Dipolar and chain-linking effects on the rheology of grafted chains in a nanopore under shear at different grafting densities.

Nonequilibrium molecular dynamics simulations are applied to investigate the rheological properties of coplanar nanopore systems of amphiphilic chain molecules with the tails grafted to the walls of the nanopore and with the head-group ends immersed in a solvent inside the nanopore. In particular, the effects of modifying the interaction between the amphiphilic head-groups by repulsive dipolar interactions or directly covalently linking pairs of chains at the head-groups have been studied. Different grafting densities are considered. The chains are modeled by a harmonic bead-spring model, and all particles interact through the repulsive part of a shifted Lennard-Jones potential. Head-group linking is also governed by a bead-spring potential. A harmonic potential models the lattice vibrations of the atomic boundaries. The rheological properties are studied by a shearing process in which the heat generated is conducted away from the system through the walls by applying a Nosé-Hoover thermostat. Computed geometric parameters such as average chain length and average tilt angle indicate reduction in chain flexibility at large dipole moments. Dipolar repulsion is found to broaden the density profiles of the solvent. This effect is opposed by chain linking. For increasing head-group repulsion, the amphiphile-solvent interfaces become less diffusive that leads to systematic variations in viscosities with increasing dipole moments. Friction forces become stronger at large grafting density and for larger dipole moments. The changes in rheological properties for fixed grafting density are essentially governed by the change in the response of the normal pressure to the applied shear field. The velocity gradients depend strongly on the degree of grafting density but appear to be less sensitive to the strength of the interactions between the head groups.

Screening effect of PEG on avidin binding to liposome surface receptors.

This study investigates the screening effect of poly(ethylene glycol)-phospholipids (PE-PEG) on the interaction of avidin with PEGylated liposomes containing surface-bound biotin ligands. The influence of grafting density and lipopolymer chain length is examined. A simple fluorescence assay involving a receptor-mediated fluorescence increase of BODIPY-labeled avidin upon binding to biotinylated lipids is employed to study the screening effect of submicellar concentrations of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[poly(ethylene glycol)-2000] (PE-PEG(2000)) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-N-[poly(ethylene glycol)-5000] (PE-PEG(5000)) incorporated into 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) liposomes. The results show that incorporation of lipopolymers into DPPC lipid bilayers reduces binding of avidin to the biotinylated liposomes, and it is found that the screening effect of PE-PEG(5000) is stronger than that for PE-PEG(2000). Thus, the results reveal that both the grafting density and the polymer length of the PE-PEG lipopolymers are of importance for the ability of water-soluble macromolecules to reach the surface of PEG liposomes. Furthermore, it is found that none of the lipopolymers completely prevents avidin from reaching the surface-bound biotin ligands.

Drug delivery by phospholipase A(2) degradable liposomes.

The effect of poly(ethylene glycol)-phospholipid (PE-PEG) lipopolymers on phospholipase A(2) (PLA(2)) hydrolysis of liposomes composed of stearoyl-oleoylphosphatidylcholine (SOPC) was investigated. The PLA(2) lag-time, which is inversely related to the enzymatic activity, was determined by fluorescence, and the zeta-potentials of the liposomes were measured as a function of PE-PEG lipopolymer concentration. A significant decrease in the lag-time, and hence an increase in enzymatic activity, was observed with increasing amounts of the negatively charged PE-PEG lipopolymers incorporated into the SOPC liposomes. The enhancement of the PLA(2) enzymatic activity might involve a stronger PLA(2) binding affinity towards the negatively charged and polymer covered PEG liposomes.

Association of an acylated model peptide with DPPC-DPPS lipid membranes.

The interaction between a small positively charged peptide with a N-terminally linked acyl chain and dipalmitoylphosphatidylcholine-dipalmitoylphosphatidylserine (DPPC-DPPS) lipid membranes has been studied by means of fluorescence resonance energy transfer. Two different lipid compositions were used: a neutral membrane (100 mol% DPPC), and a negatively charged membrane (30 mol% DPPS in DPPC). The fluorescence resonance energy transfer results reveal that the peptide associates with both types of membranes. Furthermore, it is found that the slope of the titration curve for the negatively charged membranes is much steeper than that for the neutral membranes. This indicates a higher binding affinity of the acylated peptide towards negatively charged lipid membranes as compared with neutral lipid membranes.

Lateral organization and domain formation in a two-component lipid membrane system.

The thermodynamic phase behavior and lateral lipid membrane organization of unilamellar vesicles made from mixtures of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC) were investigated by fluorescence resonance energy transfer (FRET) as a function of temperature and composition. This was done by incorporating a headgroup-labeled lipid donor (NBD-DPPE) and acceptor (N-Rh-DPPE) in low concentrations into the binary mixtures. Two instances of increased energy transfer efficiency were observed close to the phase lines in the DMPC/DSPC phase diagram. The increase in energy transfer efficiency was attributed to a differential preference of the probes for dynamic and fluctuating gel/fluid coexisting phases. This differential preference causes the probes to segregate (S. Pedersen, K. Jørgensen, T. R. Baekmark, and O. G. Mouritsen, 1996, Biophys. J. 71:554-560). The observed increases in energy transfer match with the boundaries of the DMPC/DSPC phase diagram, as measured by Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). We propose that the two instances of probe segregation are due to the presence of DMPC-rich and DSPC-rich domains, which form a dynamic structure of gel/fluid coexisting phases at two different temperatures. Monitoring the melting profile of each lipid component independently by FTIR shows that the domain structure is formed by DMPC-rich and DSPC-rich domains rather than by pure DMPC and DSPC domains.

Enzymatic degradation of polymer covered SOPC-liposomes in relation to drug delivery.

Polyethylenoxide (PEG) covered liposomes are used as lipid-based drug-delivery systems. In comparison to conventional liposomes the polymer-covered liposomes display a long circulation half-life in the blood stream. We investigate the influence of polyethyleneoxide-distearoylphosphatidylethanolamine (DSPE-PEG750) lipopolymer concentration on phospholipase A2 (PLA2) catalyzed hydrolysis of liposomes composed of stearoyloleoylphosphatidylcholine (SOPC). The characteristic PLA2 lag-time was determined by fluorescence and the degree of lipid hydrolysis was followed by HPLC analysis. Particle size and zeta-potential were measured as a function of DSPE-PEG750 lipopolymer concentration. A significant decrease in the lag-time, and hence an increase in enzyme activity, was observed with increasing concentrations of the anionic DSPE-PEG750 lipopolymer lipids. The observed decrease in lag-time might be related to changes in the surface potential and the PLA2 lipid membrane affinity.

Receptor mediated binding of avidin to polymer covered liposomes.

Fluoresence technique involving a receptor-mediated fluorescence increase of bodipy-labeled avidin upon binding to biotinylated lipids has been used to investigate the steric barrier effect of submicellar concentrations of poly(ethylene glycol)-phospholipids (PE-PEG(2000) and PE-PEG(5000)) incorporated into pure DPPC liposomes as well as PE-PEG(5000) incorporated into DPPC liposomes containing 20 mol% cholesterol. It is found that the incorporation of PE-PEG lipopolymers into DPPC lipid bilayers lowers the receptor-mediated adhesion of avidin to the biotinylated liposomes. The most pronounced screening effect is observed at surface densities corresponding to the mushroom conformation of the polymer. Furthermore, the results show that the steric baric effect induced by the surface-grafted polymers becomes stronger when the length of the polymer chain increases. In addition it is found that cholesterol improves the barrier effect of PE-PEG(5000) at low lipopolymer concentrations while no effect is observed at higher concentrations. The results reveal that both the surface density and the polymer length of the PE-PEG lipopolymers play a major role for the accessibility of avidin to biotin surface receptors. However, none of the lipopolymers were capable of completely preventing avidin from reaching the surface bound ligands. Cholesterol only affected the barrier effect at lipopolymer concentrations below the mushroom to brush transition. Consequently, from a steric stabilization viewpoint there is no rationale for incorporating cholesterol into liposomes when the PE-PEG lipopolymer concentration exceeds the mushroom to brush transition. The results presented in this study are of importance in relation to a deeper understanding of the interaction of liposome degrading enzymes and proteins with polymer covered liposomes as well as for the receptor-based targeting and interaction of liposomes with cell surface receptors.

A mathematical expectation model for bird navigation based on the clock-and-compass strategy.

We present here a mathematical formula for the directional distribution of migratory birds if they use a vector navigation/clock-and-compass strategy to find their winter quarters. It is based on mathematical expectation theory and shows that a simple parabola can describe the expected geographical spread of clock-and-compass birds as a function of migratory distance. Predictions based on this model are then tested against all same autumn ringing recoveries of first-season Pied Flycatchers, Ficedula hypoleuca, ringed in Scandinavia and European Robins, Erithacus rubecula, ringed in Sweden and Finland and recovered north of the Sahara Desert. We find that the predictions of our analytical model fit the ringing recovery distribution of freely migrating conspecifics extremely well.

Dimyristoylphosphatidylcholine/C16:0-ceramide binary liposomes studied by differential scanning calorimetry and wide- and small-angle x-ray scattering.

Ceramide has recently been established as a central messenger in the signaling cascades controlling cell behavior. Physicochemical studies have revealed a strong tendency of this lipid toward phase separation in mixtures with phosphatidylcholines. The thermal phase behavior and structure of fully hydrated binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and N-palmitoyl-ceramide (C16:0-ceramide, up to a mole fraction X(cer) = 0.35) were resolved in further detail by high-sensitivity differential scanning calorimetry (DSC) and x-ray diffraction. Both methods reveal very strong hysteresis in the thermal phase behavior of ceramide-containing membranes. A partial phase diagram was constructed based on results from a combination of these two methods. DSC heating scans show that with increased X(cer) the pretransition temperature T(p) first increases, whereafter at X(cer) > 0.06 it can no longer be resolved. The main transition enthalpy DeltaH remains practically unaltered while its width increases significantly, and the upper phase boundary temperature of the mixture shifts to approximately 63 degrees C at X(cer) = 0.30. Upon cooling, profound phase separation is evident, and for all of the studied compositions there is an endotherm in the region close to the T(m) for DMPC. At X(cer) >/= 0.03 a second endotherm is evident at higher temperatures, starting at 32.1 degrees C and reaching 54.6 degrees C at X(cer) = 0.30. X-ray small-angle reflection heating scans reveal a lamellar phase within the temperature range of 15-60 degrees C, regardless of composition. The pretransition is observed up to X(cer) < 0.18, together with an increase in T(p). In the gel phase the lamellar repeat distance d increases from approximately 61 A at X(cer) = 0. 03, to 67 A at X(cer) = 0.35. In the fluid phase increasing X(cer) from 0.06 to 0.35 augments d from 61 A to 64 A. An L(beta')/L(alpha) (ripple/fluid) phase coexistence region is observed at high temperatures (from 31 to 56.5 degrees C) when X(cer) > 0.03. With cooling from temperatures above 50 degrees C we observe a slow increase in d as the coexistence region is entered. A sudden solidification into a metastable, modulated gel phase with high d values is observed for all compositions at approximately 24 degrees C. The anomalous swelling for up to X(cer) = 0.30 in the transition region is interpreted as an indication of bilayer softening and thermally reduced bending rigidity.

A thermodynamic study of the effects of cholesterol on the interaction between liposomes and ethanol.

The association of ethanol with unilamellar dimyristoyl phosphatidylcholine (DMPC) liposomes of varying cholesterol content has been investigated by isothermal titration calorimetry over a wide temperature range (8-45 degrees C). The calorimetric data show that the interaction of ethanol with the lipid membranes is endothermic and strongly dependent on the phase behavior of the mixed lipid bilayer, specifically whether the lipid bilayer is in the solid ordered (so), liquid disordered (ld), or liquid ordered (lo) phase. In the low concentration regime (<10 mol%), cholesterol enhances the affinity of ethanol for the lipid bilayer compared to pure DMPC bilayers, whereas higher levels of cholesterol (>10 mol%) reduce affinity of ethanol for the lipid bilayer. Moreover, the experimental data reveal that the affinity of ethanol for the DMPC bilayers containing small amounts of cholesterol is enhanced in the region around the main phase transition. The results suggest the existence of a close relationship between the physical structure of the lipid bilayer and the association of ethanol with the bilayer. In particular, the existence of dynamically coexisting domains of gel and fluid lipids in the transition temperature region may play an important role for association of ethanol with the lipid bilayers. Finally, the relation between cholesterol content and the affinity of ethanol for the lipid bilayer provides some support for the in vivo observation that cholesterol acts as a natural antagonist against alcohol intoxication.

Association of ethanol with lipid membranes containing cholesterol, sphingomyelin and ganglioside: a titration calorimetry study.

The association of ethanol at physiologically relevant concentrations with lipid bilayers of different lipid composition has been investigated by use of isothermal titration calorimetry (ITC). The liposomes examined were composed of combinations of lipids commonly found in neural cell membranes: dimyristoyl phosphatidylcholine (DMPC), ganglioside (GM(1)), sphingomyelin and cholesterol. The calorimetric results show that the interaction of ethanol with fluid lipid bilayers is endothermic and strongly dependent on the lipid composition of the liposomes. The data have been used to estimate partitioning coefficients for ethanol into the fluid lipid bilayer phase and the results are discussed in terms of the thermodynamics of partitioning. The presence of 10 mol% sphingomyelin or ganglioside in DMPC liposomes enhances the partitioning coefficient by a factor of 3. Correspondingly, cholesterol (30 mol%) reduces the partitioning coefficient by a factor of 3. This connection between lipid composition and partitioning coefficient correlates with in vivo observations. Comparison of the data with the molecular structure of the lipid molecules suggests that ethanol partitioning is highly sensitive to changes in the lipid backbone (glycerol or ceramide) while it appears much less sensitive to the nature of the head group.

Off-lattice model for the phase behavior of lipid-cholesterol bilayers.

Lipid bilayers exhibit a phase behavior that involves two distinct, but coupled, order-disorder processes, one in terms of lipid-chain crystalline packing (translational degrees of freedom) and the other in terms of lipid-chain conformational ordering (internal degrees of freedom). Experiments and previous approximate theories have suggested that cholesterol incorporated into lipid bilayers has different microscopic effects on lipid-chain packing and conformations and that cholesterol thereby leads to decoupling of the two ordering processes, manifested by a special equilibrium phase, "liquid-ordered phase," where bilayers are liquid (with translational disorder) but lipid chains are conformationally ordered. We present in this paper a microscopic model that describes this decoupling phenomena and which yields a phase diagram consistent with experimental observations. The model is an off-lattice model based on a two-dimensional random triangulation algorithm and represents lipid and cholesterol molecules by hard-core particles with internal (spin-type) degrees of freedom that have nearest-neighbor interactions. The phase equilibria described by the model, specifically in terms of phase diagrams and structure factors characterizing different phases, are calculated by using several Monte Carlo simulation techniques, including histogram and thermodynamic reweighting techniques, finite-size scaling as well as non-Boltzmann sampling techniques (in order to overcome severe hysteresis effects associated with strongly first-order phase transitions). The results provide a consistent interpretation of the various phases of phospholipid-cholesterol binary mixtures based on the microscopic dual action of cholesterol on the lipid-chain degrees of freedom. In particular, a distinct small-scale structure of the liquid-ordered phase has been identified and characterized. The generic nature of the model proposed holds a promise for a unifying description for a whole series of different lipid-sterol mixtures.

Theoretical analysis of protein organization in lipid membranes.

The fundamental physical principles of the lateral organization of trans-membrane proteins and peptides as well as peripheral membrane proteins and enzymes are considered from the point of view of the lipid-bilayer membrane, its structure, dynamics, and cooperative phenomena. Based on a variety of theoretical considerations and model calculations, the nature of lipid-protein interactions is considered both for a single protein and an assembly of proteins that can lead to aggregation and protein crystallization in the plane of the membrane. Phenomena discussed include lipid sorting and selectivity at protein surfaces, protein-lipid phase equilibria, lipid-mediated protein-protein interactions, wetting and capillary condensation as means of protein organization, mechanisms of two-dimensional protein crystallization, as well as non-equilibrium organization of active proteins in membranes. The theoretical findings are compared with a variety of experimental data.