Behavior of lysozyme adsorbed onto biological liquid crystal lipid monolayer at the air/water interface

The interaction between proteins and lipids is one of the basic problems of modern biochemistry and biophysics.The purpose of this study is to compare the penetration degree of lysozyme into 1,2-diapalmitoyl-sn-glycero-3-phosphocholine(DPPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethano-lamine(DPPE) by analyzing the data of surface pressure–area(π–A) isotherms and surface pressure–time(π–T) curves.Lysozyme can penetrate into both DPPC and DPPE monolayers because of the increase of surface pressure at an initial pressure of 15 m N/m.However,the changes of DPPE are larger than DPPC,indicating stronger interaction of lysozyme with DPPE than DPPC.The reason may be due to the different head groups and phase state of DPPC and DPPE monolayers at the surface pressure of 15 m N/m.Atomic force microscopy reveals that lysozyme was absorbed by DPPC and DPPE monolayers,which leads to self-aggregation and self-assembly,forming irregular multimers and conical multimeric.Through analysis,we think that the process of polymer formation is similar to the aggregation mechanism of amyloid fibers.     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

摘要：巨囊泡作为细胞的简化模型,其分相与出芽机理及动力学规律已引起许多领域科学家的关注。在富含胆固醇的典型生物膜体系如二棕榈酰磷脂酰胆碱DPPC(2-dihexadecanoyl-rac-glycero-3phosphocholine)/二油酰磷脂酰胆碱DOPC(dioleoyl-phosphatidylcholine)/胆固醇(Chol)的三组分形成的巨囊泡作为模型,从高温退火至低温会发生相分离,形成微畴。实验中借助荧光显微镜观察生物膜体系侧向分离的相结构图。实验发现,体系各组分的不同会影响磷脂膜的相结构和膜内微畴的成长,固定 DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇参入量的增加而变大。最后运用理论进一步分析了微畴的成长机理。     

多组分磷脂巨囊泡的相结构和胆固醇对微畴成长的影响

巨囊泡作为细胞的简化模型,其相分离与出芽动力学规律已引起许多领域科学家的关注.本实验采用DPPC/DOPC/Chol的三组分形成的巨囊泡作为模型,借助荧光显微镜观察该三组分体系侧向分离的相结构图,并对微畴的成长过程作了系统的观察研究和理论分析.实验发现:从高温的均相区域淬灭到低温的分相区域,膜表面发生侧向分离形成微畴.体系内胆固醇的掺入量的多少会影响磷脂膜的相结构和膜内微畴的成长,固定DOPC/DPPC为1:1的前提下,微畴尺寸随着胆固醇掺入量的增加而变大.     

Spin-label HF-EPR of lipid ordering in cholesterol-containing membranes

Conventional oxazolidine spin-labelled lipids have the axial¹⁴N-hyperfine tensorz-axis directed along the long axis of the lipid chain. Investigation of lateral ordering of the lipids in membranes requires measurement of thex-y Zeeman anisotropy of the nonaxialg-tensor at high fields. Both the lateral and transverse ordering of the lipid chains in membranes of dimyristoyl phosphatidylcholine containing 40 mol% cholesterol in the liquid-ordered phase have been studied with 94 GHz electron paramagnetic resonance spectroscopy. This has been done by using probe amounts of phosphatidylcholine systematically spin-labelled at positionsn along the length of thesn- 2 chain [n-PCSL, 1-acyl-2-(n-(4,4-dimethyloxazolidine-N-oxyl) stearoyl)-sn-glycero-3-phosphocholine]. Nonaxial (g_xx?g_yy) anisotropy of the spin-labelled lipid chains is detected over a wide range of temperature throughout the liquid-ordered phase. The transverse profile of lateral ordering with position,n, of chain labelling follows the profile of the rigid steroid nucleus of cholesterol. It becomes progressively averaged towards the terminal methyl group of thesn- 2 chain, in the region of the flexible hydrocarbon chain of cholesterol. The nonaxial lipid ordering may be related to lipid domain formation in membranes containing cholesterol and saturated-chain lipids.     

Miscibility phase diagrams of giant vesicles containing sphingomyelin

Saturated sphingomyelin (SM) lipids are implicated in lipid rafts in cell plasma membranes. Here we use fluorescence microscopy to observe coexisting liquid domains in vesicles containing SM, an unsaturated phosphatidylcholine lipid (either DOPC or POPC), and cholesterol. We note similar phase behavior in a model membrane mixture without SM (DOPC/DPPC/Chol), but find no micron-scale liquid domains in membranes of POPC/PSM/Chol. We delineate the onset of solid phases below the miscibility transition temperature, and detail indirect evidence for a three-phase coexistence of one solid and two liquid phases.     

Suppression and enhancement of non-native molecules within biological systems

With the aim of evaluating the potential of SIMS to provide molecular information from small molecules within biological systems, here we investigate the effect of different biological compounds as they act as matrices. The results highlight the fact that the chemical environment of a molecule can have a significant effect on its limit of detection. This has implications for the imaging of drugs and xenobiotics in tissue sections and other biological matrices.A 1:1 mixture of the organic acid 2,4,6-trihydroxyacetophenone and the dipeptide valine-valine demonstrates that almost complete suppression of the [M + H]⁺ ion of one compound can be caused by the presence of a compound of higher proton affinity. The significance of this is highlighted when two similar drug molecules, atropine (a neutral molecule) and ipratropium bromide (a quaternary nitrogen containing salt) are mixed with brain homogenate. The atropine [M + H]⁺ ion shows significant suppression whilst the [M − Br]⁺ of ipratopium bromide is detected at an intensity that can be rationalised by its decreased surface concentration.By investigating the effect of two abundant tissue lipids, cholesterol and dipalmitoylphosphatidyl choline (DPPC), on the atropine [M + H]⁺ signal detected in mixtures with these lipids we see that the DPPC has a strong suppressing effect, which may be attributed to gas phase proton transfer.     

Organization in lipid membranes containing cholesterol

A fundamental attribute of raft formation in cell membranes is lateral separation of lipids into coexisting liquid phases. Using fluorescence microscopy, we observe spontaneous lateral separation in free-floating giant unilamellar vesicles. We record coexisting liquid domains over a range of composition and temperature significantly wider than previously reported. Furthermore, we establish correlations between miscibility in bilayers and in monolayers. For example, the same lipid mixtures that produce liquid domains in bilayer membranes produce two upper miscibility critical points in the phase diagrams of monolayers.     

Role of non-lamellar-forming lipid in promotion of liposomal fusion

Membrane fusion is an important process in a wide range of cellular and sub-cellular activities. It is evident that during the intermediate stages of fusion some transitory non-bilayer configurations must appear within the lipid moiety. Using fluorescence techniques, we have studied here the process of aggregation and fusion of liposomes made of lipids, namely 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). When mixed together, the complete fusion between these two liposomes took around 44 h as both DPPC and DMPC favour lamellar configuration. When the mixture was incubated at 42°C the fusion process was completed after 23 h. But, when 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) was added in the liposomal matrix the time for fusion was reduced to 21 h for mixture without incubation and 17 h when the mixture was incubated. This indicates that DPPE having a tendency to assume non-lamellar conformation, promoted destabilisation of the lamellar conformation within the liposome which facilitated the fusion between two apposing bilayers.     

Temperature effect on thin lipid film elasticity and phase separation: insights from Langmuir monolayer and fluorescence microscopy techniques

Langmuir monolayer pressure isotherms and compressibility modulus measurements of phospholipid mixtures in several Langmuir monolayer systems at the air/water interface were investigated in this study. The ultimate aim was to carry out a comparison of the elasticity modulus for monolayers with different mixtures of l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and chicken egg yolk sphingomyelin (eSM), in the presence/absence of cholesterol (Chol). In particular, we were able to propose that the leading force beyond the phase separation into liquid expanded (LE-) and liquid condensed (LC-) phases emerges from the increasing barrier to incorporate DOPC molecules into a highly ordered LC-phase. In addition, our findings suggest that DOPC lipid molecules have a priority to incorporate in a disordered LE-phase, while DPPC and eSM prefer the ordered one. Also, Chol seems to split almost equally into both phases, indicating that Chol has no priority for either phase and there are no particular interactions between Chol and saturated lipid molecules.     

Patterning silver nanocubes in monolayers using phase separated lipids as templates

Strategies for assembling silver nanocubes (NCs) into distinct 2D patterns on Langmuir–Blodgett (LB) films are demonstrated using two different lipid mixtures as vehicles: (1) raft mixtures containing 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SPM), and cholesterol in different mole ratios (2:2:1 and 1:1:1) and (2) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC) at a 1:3 mol ratio. Atomic force microscopy was employed to unveil the mechanisms of such pattern formation in the LB film. The results demonstrate that aggregation of NCs into round-like pattern is governed by preferential localization of NCs within the liquid condensed (LC) domains of DOPC/SPM/Cholesterol mixture. Cholesterol was found to govern the size and shape of the rounded islands. On the other hand, incorporation of NCs within the liquid expanded (LE) phase of DPPC/DLPC mixture produced linear-branched chains, oriented normal to the Langmuir film transfer direction. The as engineered patterns of silver NCs exhibited characteristic plasmonic signatures. Our results reveal the potential in assembling plasmonic metal nanoparticles into diverse patterns on solid substrates by exploiting their preferential localization either in LC or LE phase of appropriate lipid mixture in Langmuir film.     

Influence of membrane lipid composition on the activity of functionally reconstituted LmrA under high hydrostatic pressure

In cellular membranes, proteins and lipids are in sensitive macromolecular interaction influencing each other. To evaluate this interaction, the multi-drug transporter LmrA from Lactococcus lactis was functionally reconstituted in vesicles consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), DMPC+10 mol% cholesterol and the model raft mixture DOPC/1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (1:2:1) and in natural membrane lipids at 30 °C. The lateral structure and organization of these proteoliposomes were modulated using high hydrostatic pressure. A sharp pressure-induced fluid-to-gel phase transition is observed without an extended two-phase region. The possibility for lipid sorting, such as for DMPC/cholesterol bilayers, has an inhibitory effect on the LmrA activity. A fluid-like membrane phase over the whole pressure range with suitable hydrophobic matching, such as for DOPC, prevents the membrane protein from high-pressure inactivation up to 200 MPa. Under high-pressure conditions, highest LmrA activities, exceeding those at ambient pressure, are achieved for heterogeneous lipid matrices with a small hydrophobic mismatch and the ability of lipid sorting.     

Lateral diffusion in equimolar mixtures of natural sphingomyelins with dioleoylphosphatidylcholine

Cellular membranes of mammals are composed of a complex assembly of diverse phospholipids. Sphingomyelin (SM) and phosphatidylcholine (PC) are important lipids of eukaryotic cellular membranes and neuronal tissues, and presumably participate in the formation of membrane domains, known as "rafts," through intermolecular interaction and lateral microphase decomposition. In these two-dimensional membrane systems, lateral diffusion of lipids is an essential dynamic factor, which might even be indicative of lipid phase separation process. Here, we used pulsed field gradient nuclear magnetic resonance to study lateral diffusion of lipid components in macroscopically oriented bilayers composed of equimolar mixtures of natural SMs of egg yolk, bovine brain, bovine milk and dipalmitoylphosphatidylcholine (DPPC) with dioleoylphosphatidylcholine (DOPC). In addition, differential scanning calorimetry was used as a complementary technique to characterize the phase state of the lipid bilayers. In fully liquid bilayers, the lateral diffusion coefficients in both DOPC/DPPC and DOPC/SM systems exhibit mean values of the pure bilayers. For DOPC/SM bilayer system, this behavior can be explained by a model where most SM molecules form short-lived lateral domains with preferential SM-SM interactions occurring within them. However, for bilayers in the presence of their low-temperature gel phase, lateral diffusion becomes complicated and cannot simply be understood solely by a simple change in the liquid phase decomposition.     

Thermodynamic approach to phase coexistence in ternary phospholipid-cholesterol mixtures

We introduce a simple and predictive model for determining the phase stability of ternary phospholipid-cholesterol mixtures. Assuming that competition between the liquid and gel order of the phospholipids is the main driving force behind lipid segregation, we derive a Gibbs free energy of mixing, based on the thermodynamic properties of the lipids main transition. A numerical approach was devised that enables the fast and efficient determination of the ternary diagrams associated with our Gibbs free energy. The computed phase coexistence diagram of DOPC/DPPC/cholesterol reproduces well-known features for this system at 10 °C, as well as its evolution with temperature.     

A new theoretical approach to study the effects of active molecules on lipid bilayer properties: The cholesterol problem

A model to study phospholipid bilayers with active molecules that greatly disturb molecular conformations of their neighboring lipids is proposed. In the particular case of cholesterol in DPPC bilayers, this method allows us to use the spin 1 Ising hamiltonian as a natural extension of the two state model of chain melting transition. Physical processes responsible for the decreasing of the total transition enthalpy Δ_tQ and temperature T_c, and for the phase separation, both below and above T_c, when cholesterol is added, are easily identified. From the details of the phase separation, the two peak specific heat data are explained. Similar models for the addition of other binding molecules to lipid bilayers are discussed.     

Packing of phospholipid vesicles studied by oxygen quenching of Laurdan fluorescence

Steady-state fluorescence oxygen quenching experiments were performed on phospholipid vesicles where 2-dimethylamino-6-lauroylnaphthalene (Laurdan) was inserted. The quenching efficiency was found to be much higher in vesicles in the liquid-crystalline phase with respect to the gel phase, by a factor of about 50. Since the oxygen solubility in the two phospholipid phases can differ at most by a factor of 4 based on literature values, we concluded that oxygen diffusion must be responsible for the great difference in the quenching efficiency. A relatively high quenching efficiency was also found in vesicles composed of equimolar gel and liquid-crystalline phospholipids. Simulations were performed using the linear superposition of the properties of the pure phases to demonstrate that, in the case of vesicles composed of coexisting phases, the diffusional properties of oxygen in each phase are largely modified by the presence of the other. The addition of 10 mol% cholesterol to the gel phase rendered Laurdan fluorescence approximately as quenchable as in the equimolar mixture of the two phases. This result points out that molecules such as cholesterol, which introduce packing defects in the bilayer, favor oxygen diffusion. From the oxygen quenching experiments and using the properties of generalized polarization, the rate of Laurdan dipolar relaxation can be estimated.Abbreviations used Laudran 2-dimethylamino-6-lauroylnapthalene - DLPC dilauroylphosphatidylcholine - DMPC dimyristoylphosphatidylcholine - DPH 1,6-diphenyl-1,3,5-hexatriene - DPPC dipalmitoylphosphatidylcholine - TNS p-tofuidinyl-6-naphthalene sulfonic acid - PBS phosphate-buffered saline solution - GP generalized polarization - NMR nuclear magnetic resonance - EPR electron paramagnetic resonance     

Phase diagram of a ternary mixture of cholesterol and saturated and unsaturated lipids calculated from a microscopic model

We employ a molecular model to study a ternary mixture of saturated lipid, with tails of 16 carbons, a monounsaturated lipid with tails of 18 carbons, and cholesterol. The model, solved within mean-field theory, produces several forms of phase diagrams depending upon the relative strengths of interactions, but only one that shows the coexistence of two liquid phases observed in experiment. The lipids in the phase rich in cholesterol are more ordered than those in the other. The binary cholesterol, saturated lipid system also exhibits liquid, liquid coexistence.     

Influence of salicylic acid on the biophysical properties of dipalmitoyl phosphatidylcholine vesicles

The effect of the keratolytic drug salicylic acid (SA) on the thermotropic behaviour, and dynamics of dipalmitoyl phosphatidyl choline (DPPC)–water/buffer pH?7.4 vesicles was studied using DSC and ¹H NMR. In both systems, incorporation of SA in DPPC bilayer causes a significant depression in the transition temperature of both the pre-transition (PT) and the gel-to-liquid crystalline (CM) transition. The presence of the drug reduces the cooperativity of both the PT and CM transitions. These findings indicate that SA is bound strongly to the lipid bilayer leading to increased membrane fluidity. The DPPC vesicles incorporated with high drug concentration show phase segregation. One of the interesting findings in this study is the formation of a more ordered high temperature gel (L_β2) phase when the SA-doped DPPC dispersion is prepared at physiological pH. The effect of inclusion of cholesterol in the SA-free and SA-doped DPPC dispersion was also studied.     

Raman and X‐ray investigations of the incorporation of Ca2+ and Cd2+ in the ZrO2 structure

The formation of solid solution and ZrO₂ phase stabilization were investigated by Raman spectroscopy and X‐ray diffraction (XRD) in calcium‐containing and cadmium‐containing zirconium oxide samples heated at 1073 K in air. The adopted preparation procedure led to the incorporation of calcium and cadmium in solid solution into the zirconia structure. The solid solution favored the tetragonal and cubic zirconia phases at the expense of the thermodynamically stable monoclinic modification. Combined macro‐ and micro‐Raman spectroscopy disclosed that instead of forming a homogeneous phase t″, intermediate between the tetragonal t′ and the cubic phase, the tetragonal and cubic phases coexisted in the range 9.49–13.89 mol% for Ca and 11.88–17.23 mol% for Cd. At higher dopant contents the cubic form stabilized. The impurity content necessary to stabilize the high‐symmetry phases was defined. Copyright © 2007 John Wiley & Sons, Ltd.     

A simulation study on multicomponent lipid bilayer

Simulation of a multicomponent lipid bilayer having a fixed percentage of cholesterol is done to study phase transition leading to domain formation. The concept of random lattice has been used in simulation to account for the coupling between the internal and translational degrees of freedom of lipid molecules. Considering a canonical ensemble, dissimilar lipid molecules are allowed to exchange their positions in the lattice subject to standard metropolis algorithm. The steps involved in the process effectively takes into account for the movement of sphingolipids and cholesterol molecules helping formation of cholesterol rich domains of saturated lipids as found in natural membranes.     

Thermal conductivities of solid and liquid phases for neopentylglycol, aminomethylpropanediol and their binary alloy

The variations of thermal conductivities of solid phases versus temperature for neopentylglycol (NPG), 2-amino-2-methyl-1,3-propanediol (AMPD) and AMPD-42.2 mol% NPG alloy were measured with a radial heat flow apparatus. From the graphs of the solid phases thermal conductivity variations versus temperature, the thermal conductivities of the solid phases at their melting temperature and temperature coefficients for same materials were also found to be 0.22±0.01, 0.45±0.02 and 0.32±0.02 W/Km and 0.0047, 0.0031 and 0.0043 K⁻¹, respectively. The thermal conductivity ratios of liquid phase to solid phase for the same materials at their melting temperature are found to be 1.07, 1.12 and 0.74 with a Bridgman type directional solidification apparatus, respectively. Thus, the thermal conductivities of liquid phases for pure NPG, pure AMPD and AMPD-42.2 mol% NPG alloy at their melting temperature were evaluated to be 0.24, 0.50 and 0.23 W/Km, respectively, by using the values of solid phase thermal conductivities and the thermal conductivity ratios of liquid phase to solid phase.