Wetting and capillary condensation as means of protein organization in membranes.

Wetting and capillary condensation are thermodynamic phenomena in which the special affinity of interfaces to a thermodynamic phase, relative to the stable bulk phase, leads to the stabilization of a wetting phase at the interfaces. Wetting and capillary condensation are here proposed as mechanisms that in membranes may serve to induce special lipid phases in between integral membrane proteins leading to long-range lipid-mediated joining forces acting between the proteins and hence providing a means of protein organization. The consequences of wetting in terms of protein aggregation and protein clustering are derived both within a simple phenomenological theory as well as within a concrete calculation on a microscopic model of lipid-protein interactions that accounts for the lipid bilayer phase equilibria and direct lipid-protein interactions governed by hydrophobic matching between the lipid bilayer hydrophobic thickness and the length of the hydrophobic membrane domain. The theoretical results are expected to be relevant for optimizing the experimental conditions required for forming protein aggregates and regular protein arrays in membranes.     

Modelling the phase equilibria in two-component membranes of phospholipids with different acyl-chain lengths

A phenomenological model is proposed to describe the membrane phase equilibria in binary mixtures of saturated phospholipids with different acyl-chain lengths. The model is formulated in terms of thermodynamic and thermomechanic properties of the pure lipid bilayers, specifically the chain-melting transition temperature and enthalpy, the hydrophobic bilayer thickness, and the lateral area compressibility modulus. The model is studied using a regular solution theory made up of a set of interaction parameters which directly identify that part of the lipid-lipid interaction which is due to hydrophobic mismatch of saturated chains of different lengths. It is then found that there is effectively a single universal interaction parameter which, in the full composition range, describes the phase equilibria in mixtures of DMPC/DPPC, DPPC/DSPC, DMPC/DSPC, and DLPC/DSPC, in excellent agreement with experimental measurements. The model is used to predict the variation with temperature and composition of the specific heat, as well as of the average membrane thickness and area in each of the phases. Given the value of the universal interaction parameter, the model is then used to predict the phase diagrams of binary mixtures of phospholipids with different polar head groups, e.g., DPPC/DPPE, DMPC/DPPE and DMPE/DSPC. By comparison with experimental results for these mixtures, it is shown that difference in acyl-chain lengths gives the major contribution to deviation from ideal mixing. Application of the model to mixtures with non-saturated lipids is also discussed.     

Lipid membrane domain formation and alamethicin aggregation studied by calorimetry, sound velocity measurements, and atomic force microscopy

An experimental study of phosphocholine membranes made from one lipid, from mixtures of DPPC and DLPC, and also from lipids and small amounts of alamethicin is presented. We used atomic force microscopy to investigate the spatial organization and structure of lipid domains and also of the defects induced by the peptide. Alamethicin was found to alter the state of lipids in the gel state in a way that domains of fluid lipids are formed close to the defects. Differential calorimetry revealed phase characteristics of the lipid mixtures and the effect of small amounts of alamethicin on the phase behavior. It was also shown that the sound velocity profiles of the membranes suspensions can be well calculated from the heat capacity traces of the samples. This result confirms the correlation between the mechanical properties and the specific heat of membrane systems.     

The kinetics and thermodynamics of bleaching of rhodopsin in dimyristoylphosphatidylcholine. Identification of meta-I, meta-II, and meta-III intermediates.

The effects of light on rhodopsin reconstituted into dimyristoylphosphatidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stability of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibria, whereas lipid-protein interactions were also directly involved. Rhodopsin photolysis resulted in temperature-sensitive equilibria between three main photoproducts, with absorption maximal of approximately 480, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was formed. The photoproduct with a spectral-maximum at 465 nm absorption was formed more slowly. Increasing temperatures decreased the ratio of the 465:380 nm-absorbing species. The thermal reactions were reversible: on cooling the higher-temperature products were converted back to the lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyristoylphosphatidylcholine. The equilibria that we have measured resemble those of rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstituted membranes and the meta-I-to-meta-II equilibrium is displaced in the direction of the meta-I state relative to native rod outer segment disk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the effects of lipid-lipid interactions in the membrane bilayer and correlates with the difference in gel-to-fluid phase transition temperature of the different lipids.     

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.     

Voltage-induced capacitance relaxation of lipid bilayer membranes Effects of membrane composition

The specific capacity of black lipid membranes of different phospholipids dissolved in n-alkanes was investigated. The hydrocarbon thickness of these membranes, as calculated from the electric capacity with a dielectric constant of 2.1, was in most cases close to 5 nm. It was found that the specific capacity is not constant with time after blackening. It shows a linear time dependence characteristic for each lipid/solvent system.The influence of a transmembrane potential on the capacity of the membranes was measured. It was shown that the extent of the capacity change, obtained 10 s after applying a voltage, was strongly dependent on the lipid composition as well as the solvent content of the membranes. The capacity change of the membranes seems to be cause mainly by a thickness change and not by an area increase of the membranes.     

The effects of density fluctuations on the partitioning of foreign molecules into lipid bilayers: application to anaesthetics and insecticides

An extensive computer-simulation study is performed on a simple but general molecular model recently proposed (J?rgensen et al. (1991) Biochem. Biophys. Acta 1062, 277-238) to describe foreign molecules interacting with lipid bilayers. The model is a multi-state lattice model of the main bilayer transition in which the foreign molecules are assumed to intercalate at interstitial lattice positions. Specific as well as non-specific interactions between the foreign molecules and the lipid acyl chains are considered. Particular attention is paid to the fluctuating properties of the membrane and how the presence of the foreign molecules modulates these fluctuations in the transition region. By means of computer-stimulation techniques, a detailed account is given of the macroscopic as well as microscopic consequences of the fluctuations. The macroscopic consequences of the fluctuations are seen in the thermal anomalies of the specific heat and the passive trans-membrane permeability. Microscopically, the fluctuations manifest themselves in lipid-domain formation in the transition region which implies an effective dynamic membrane heterogeneity. Within the model it is found that certain anaesthetics and insecticides which are characterised by specific interactions with the lipids have a strong effect on the heterogeneity of the membrane inducing regions of locally very high concentration of the foreign molecules. This leads to a broadening of the specific heat peak and a maximum in the membrane/water partition coefficient. These results are in accordance with available experimental data for volatile general anaesthetics like halothane, local anaesthetics like cocain derivatives, and insecticides like lindane.     

Phase equilibria in the phosphatidylcholine-cholesterol system

A thermodynamic and a microscopic interaction model are proposed to describe the phase equilibria in the phosphatidylcholine-cholesterol system. The model calculations allow for a solid phase with conformationally ordered acyl chains and liquid phases with conformationally ordered as well as disordered chains. The resulting phase diagram is in excellent agreement with the experimental phase diagram for dipalmitoylphosphatidylcholine bilayers with cholesterol as determined by a recent NMR and calorimetry study. It is thus demonstrated that the phase behaviour of phosphatidylcholine-cholesterol mixtures can be rationalized using only a few basic assumptions: (i) Cholesterol interacts favourably with phosphatidylcholine chains in an extended conformation, (ii) the main transition of pure phosphatidylcholine bilayers takes place in terms of translational variables as well acyl-chain conformational variables, and (iii) cholesterol disturbs the translational order in the crystalline (gel) state of phosphatidylcholine. These results suggest that the occurrence of specific phosphatidylcholine-cholesterol complexes is not implied by the experimental thermodynamic data.     

The fats of Escherichia coli during infancy and old age: regulation by global regulators, alarmones and lipid intermediates

The fluidity and phase state of bacterial lipid bilayers commonly change in response to ambient environmental conditions to maintain the critical functions of the envelope as a semipermeable and selective boundary. A special, and intricate, set of alterations in membrane lipid metabolism is elicited by conditions causing growth arrest. Under such conditions, specific alterations in the membrane lipid–fatty acid composition are required for survival of the cell and, concurrently, the membrane lipids are suggested to serve as endogenous reserves providing carbon/energy for maintenance requirements. It appears that the global regulator FadR is required for both of these activities to be performed properly and that the FadR regulon is interconnected to the universal stress response of Escherichia coli . FadR, in conjuction with long-chain fatty acyl-CoA, long-chain acyl-ACP, ppGpp and cAMP, are key players in regulating the activities of enzymes and expression of genes involved in fatty acid and phospholipid metabolism in dividing and ageing E . coli cells.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by 31P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing. In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for 31P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 degrees C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 degrees C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Numerical study of the phase separation in binary lipid membrane containing protein inclusions under stationary shear flow

The phase separation of lipids is believed to be responsible for the formation of lipid rafts in biological cell membrane. In the present work, a continuum model and a particle model are constructed to study the phase separation in binary lipid membrane containing inclusions under stationary shear flow. In each model, employing the cell dynamical system (CDS) approach, the kinetic equations of the confusion-advection process are numerically solved. Snapshot figures of the phase morphology are performed to intuitively display such phase evolving process. Considering the effects from both the inclusions and the shear flow, the time growth law of the characteristic domain size is discussed.     

Equilibrium analysis and phase synchronization of two coupled HR neurons with gap junction

The properties of equilibria and phase synchronization involving burst synchronization and spike synchronization of two electrically coupled HR neurons are studied in this paper. The findings reveal that in the non-delayed system the existence of equilibria can be turned into intersection of two odd functions, and two types of equilibria with symmetry and non-symmetry can be found. With the stability and bifurcation analysis, the bifurcations of equilibria are investigated. For the delayed system, the equilibria remain unchanged. However, the Hopf bifurcation point is drastically affected by time delay. For the phase synchronization, we focus on the synchronization transition from burst synchronization to spike synchronization in the non-delayed system and the effect of coupling strength and time delay on spike synchronization in delayed system. In addition, corresponding firing rhythms and spike synchronized regions are obtained in the two parameters plane. The results allow us to better understand the properties of equilibria, multi-time-scale properties of synchronization and temporal encoding scheme in neuronal systems.     

Phase behavior of phosphatidylglycerol in spinach thylakoid membranes as revealed by P-NMR

Non-bilayer lipids account for about half of the total lipid content in chloroplast thylakoid membranes. This lends high propensity of the thylakoid lipid mixture to participate in different phases which might be functionally required. It is for instance known that the chloroplast enzyme violaxanthin de-epoxidase (VDE) requires a non-bilayer phase for proper functioning in vitro but direct evidence for the presence of non-bilayer lipid structures in thylakoid membranes under physiological conditions is still missing.In this work, we used phosphatidylglycerol (PG) as an intrinsic bulk lipid label for ³¹P-NMR studies to monitor lipid phases of thylakoid membranes. We show that in intact thylakoid membranes the characteristic lamellar signal is observed only below 20 °C. But at the same time an isotropic phase is present, which becomes even dominant between 14 and 28 °C despite the presence of fully functional large membrane sheets that are capable of generating and maintaining a transmembrane electric field. Tris-washed membranes show a similar behavior but the lamellar phase is present up to higher temperatures. Thus, our data show that the location of the phospholipids is not restricted to the bilayer phase and that the lamellar phase co-exists with a non-bilayer isotropic phase.     

Effect of valinomycin on the structure of water-lecithin liposomes]

It has been established that water--lecithin liposomes in the heptane phase are formed in the course of equilibration of lecithin solution in heptane with water phase containing Na, K-picrate. The salts penetrate both the water nucleus of liposomes and their lecithin shell. The addition of valinomycin to this system does not change the water content in liposomes, but considerably increases the salt absorbed by the lipid shell of liposomes. A characteristic S-shape curve showing the relation between picrate extraction and the valinomycin concentration might be explained as an indication of a cooperative change of the structure of lecithin shell. This phase transition is induced by one valinomycin molecule per 10(3)--10(4) lecithin molecules.     

Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

Porphyrin-membrane interactions: binding or partition?

Porphyrins are photodynamic drugs employed in an experimental tumor-treatment modality in which cell membranes are one of the primary drug-action sites. To gain insight into the nature of the interaction of these drugs with those primary sites we have studied the affinity of porphyrins to the lipid moieties of biological membranes, at the molecular level. The association of porphyrins to large unilamellar liposomes, modeling the lipid regions of biological membranes was studied (at equilibrium) for deuteroporphyrin IX and protoporphyrin IX, at neutral pH and 37 degrees C, taking into account porphyrin aggregation. Two thermodynamic approaches were investigated: (i) Simple partition equilibria between the external aqueous phase and the lipid bilayer, for drug monomers and dimers. (ii) Binding equilibria of drug monomers and dimers to the lipid bilayer. Using two types of experimental design and processing the data according to the expectations of both approaches, three different models for the binding (differing in the participation assigned to the dimer) were considered. Our major findings are: (a) The data clearly do not fit with the expectations for simple partition equilibria, nor with binding models assuming direct participation of the dimers. (b) The data fit well with a binding process, in which the membrane binds the porphyrin monomers only, with the dimers participating indirectly through the aqueous dimerization equilibrium. (c) At 37 degrees C and neutral pH, for liposomes composed of phosphatidylcholine/cholesterol at molar ratios of 3:2, we found for both investigated species a binding constant of 2.3 x 10(4) M-1. (d) For each species the binding constant is independent of the initial and final states of drug aggregation in the aqueous phase.     

Evidence for lipid involvement in the high-affinity interaction between cimetidine and rat liver cytochrome P-450

The spectral interaction between cimetidine and a cytochrome P-450 fraction isolated from liver of untreated rats has been shown to be markedly affected by dilauroylphosphatidylcholine. In the absence of the lipid the pigment preparation yielded a binding curve characteristic of a single isoenzyme species with low affinity for the drug, whereas its inclusion led to the observation of a much stronger interaction with a dissociation constant close to that obtained for the high-affinity component(s) of the parent microsomes; material with lower affinity was also observed. Gel chromatography and partition equilibrium studies yielded results which precluded interpretation of this finding either in terms of incomplete incorporation of the pigment into the phospholipid or of disproportionate solvation of cimetidine into the lipid phase of the reconstituted phospholipid complex. In contrast, phospholipid caused only a minor change in the strength of cimetidine binding by the predominant liver cytochrome P-450 from phenobarbitone-pretreated rats. Pronounced lipid sensitivity of cimetidine-binding affinity is thus not a general feature of the microsomal cytochrome P-450 system but rather a specific characteristic of individual isoenzyme species.