Influence of (phospho)lipases on properties of mica supported phospholipid layers

The effect of enzymes: lipase from Candida cylindracea (L_Cc), phospholipase A₂ from hog pancreas (PLA₂) and phospholipase C from Bacillus cereus (PLC) to modulate wetting properties of solid supported phospholipid bilayers was studied via advancing and receding contact angle measurements of water, formamide and diiodomethane, and calculation of the surface free energy and its components from van Oss et al. (LWAB) and contact angle hysteresis (CAH) approaches. Simultaneously, topography of the studied layers was determined by Atomic Force Microscopy (AFM). The investigated lipid bilayers were transferred on mica plates from subphase of pure water by means of Langmuir-Blodgett and Langmuir-Schaefer techniques. The investigated phospolipid layers were: saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and their mixture DPPC/DOPC. The obtained results revealed that the lipid membrane degradation by the enzymes caused increase in its surface free energy due to the amphiphilic hydrolysis products, which may accumulate in the lipid bilayer. In result activity of the enzymes may increase and then break down the bilayer structure takes place. It is likely that after dissolution of the hydrolysis reaction products in the bulk phase, patches of bare mica surface are accessible, which contribute to the apparent surface free energy changes. Comparison of AFM images and the free energy changes of the layers gives better insight into changes of their properties. The observed gradual increase in the layer surface free energy allows controlling of the hydrolysis process to obtain the surfaces of defined properties.     

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.     

Laurdan in Fluid Bilayers: Position and Structural Sensitivity

Laurdan (2-dimethylamino-6-lauroylnaphthalene) is a hydrophobic fluorescent probe widely used in lipid systems. This probe was shown to be highly sensitive to lipid phases, and this sensitivity related to the probe microenvironment polarity and viscosity. In the present study, Laurdan was incorporated in 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DPPG), which has a phase transition around 41°C, and DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine), which is in the fluid phase at all temperatures studied. The temperature dependence of Laurdan fluorescent emission was analyzed via the decomposition into two gaussian bands, a short- and a long-wavelength band, corresponding to a non-relaxed and a water-relaxed excited state, respectively. As expected, Laurdan fluorescence is highly sensitive to DPPG gel–fluid transition. However, it is shown that Laurdan fluorescence, in DLPC, is also dependent on the temperature, though the bilayer phase does not change. This is in contrast to the rather similar fluorescent emission obtained for the analogous hydrophilic probe, Prodan (2-dimethylamino-6-propionylnaphthalene), when free in aqueous solution, over the same range of temperature. Therefore, Laurdan fluorescence seems to be highly dependent on the lipid bilayer packing, even for fluid membranes. This is supported by Laurdan fluorescence anisotropy and spin labels incorporated at different positions in the fluid lipid bilayer of DLPC. The latter were used both as structural probes for bilayer packing, and as Laurdan fluorescence quenchers. The results confirm the high sensitivity of Laurdan fluorescence emission to membrane packing, and indicate a rather shallow position for Laurdan in the membrane.     

Changes in wetting and energetic properties of glass caused by deposition of different lipid layers

An investigation of wetting and energetic properties of different lipid layers deposited on the glass surface was carried out by contact angles measurements and determination of the apparent surface free energy. The topography of the lipid layers was also determined with the help of atomic force microscopy (AFM). Two synthetic phospholipids were chosen for these studies, having the same phosphatidylcholine headgroup bound to the apolar part composed either by two saturated chains (1,2-dipalmitoyl-sn-glycero-3-phospshocholine - DPPC) or two unsaturated chains (1,2-dioleoyl-sn-glycero-3-phosphocholine - DOPC) and one lipid (1,2,3-trihexadecanoyl-sn-glycerol - tripalmitoylglycerol - TPG). The lipid layers, from the 1st to the 5th statistical monolayer, were deposited on the glass surface from chloroform solutions by spreading.The apparent surface free energy of the deposited layers was determined by contact angles measurements (advancing and receding) for three probe liquids (diiodomethane, water, and formamide), and then two concepts of interfacial interactions were applied. In the contact angle hysteresis approach (CAH) the apparent total surface free energy was calculated from the advancing and receding contact angles and surface tension of probe liquids. In the Lifshitz-van der Waals/acid-base approach (LWAB) the total surface free energy was calculated from the determined components of the energy, which were obtained from the advancing contact angles of the probe liquids only. Comparison of the results obtained by two approaches provided more information about the changes in the hydrophobicity/hydrophilicity of the layers depending on the number of monolayers and kind of the lipid deposited on the glass surface.It was found that the most visible changes in the surface free energy took place for the first two statistical monolayers irrespectively of the kind of the lipid used. Additionally, in all cases periodic oscillations from layer-to-layer in the lipid surface free energy were observed. The changes in the surface free energy correlated with those in the topography and roughness of lipid layers.     

Resolving domains of interdigitated phospholipid membranes with 95 GHz spin labeling EPR

High-field W-band (95 GHz) electron paramagnetic resonance (EPR) study of partitioning of a small nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy) in multilamellar liposomes composed from 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) is described. The high-resolution spectra with a high signal-to-noise ratio were combined with automated least-squares simulation analysis to derive accurate partitioning coefficients of TEMPO in the membrane lipid phase and to follow the membrane phase transitions. The isotropic magnetic parameters, g_iso and A_iso were used to characterize the average polarity the spin label is experiencing in the membrane. We also report an empirical correlation between g_iso and A_iso for a set of protic and aprotic solvents and use this correlation to assign domains formed by interdigitation of DPPC bilayer under a high ethanol concentration of 1.2 M.     

A Versatile Method for Determining the Molar Ligand-Membrane Partition Coefficient

A novel method for the quantitative assessment of the membrane partitioning of a ligand from the aqueous phase is described, demonstrated here with the thoroughly studied antipsychotic chlorpromazine (CPZ). More specifically, collisional quenching of the fluorescence of a pyrene labeled fluorescent lipid analog 1-palmitoyl-2[10-(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC) by CPZ was utilized, using 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine and -serine (POPC and POPS) liposomes as model membranes. The molar partition coefficient is obtained from two series of titrations, one with constant [phospholipid] and increasing [drug] and the other with constant [drug] and varying total [phospholipid], the latter further comprising of large unilamellar vesicles (LUVs) of POPC/POPS/PPDPC at a constant concentration of 10 μM and indicated concentrations of POPC/POPS LUVs. Notably, the approach described is generic and can be employed in screening for the membrane partitioning of compounds, providing that a suitable fluorescence parameter can be incorporated into one population of liposomes utilized as model membranes.     

Damage effects of γ-radiation on lipid bilayers

Summary The lipid composition of multi-lamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine exposed to¹³⁷Cs γ-rays depends on the absorbed dose. In fact,³¹P and¹H NMR analysis shows that four new molecular species are formed during the irradiation:a) 1-palmitoyl-sn-glycero-3-phosphorylcholine,b) 2-palmitoyl-sn-glycero-3-phosphorylcholine,c) glycerophosphorylcholine andd) free palmitic acid. Neglecting the speciesc), that is present only at high dose and in very small amount, the behaviour of molar fractionvs. dose is sublinear fora) andb), while ford) it is almost linear over all the dose range examined. The molecular and structural damage consequences onto the multi-lamellar vesicles, evidentiated by spin-labelling and DSC techniques, are discussed. It is clearly shown in particular, that the behaviour of the main transition does not depend on the concentration of the lysolecithins, but rather on that of the free palmitic acid, the role of which had previously been entirely neglected. The authors of this paper have agreed to not receive the proofs for correction.     

Low-Temperature Dynamical Transition in Lipid Bilayers Detected by Spin-Label ESE Spectroscopy

Data on neutron scattering in biological systems show low-temperature dynamical transition between 170 and 230 K manifesting itself as a drastic increase of the atomic mean-squared displacement, 〈x²〉, detected for hydrogen atoms in the nano- to picosecond time scale. For spin-labeled systems, electron spin echo (ESE) spectroscopy—a pulsed version of electron paramagnetic resonance—is also capable of detection of dynamical transition. A two-pulse ESE decay in frozen matrixes is induced by spin relaxation arising from stochastic molecular librations, and allows to obtain the 〈α²〉τ_c parameter, where 〈α²〉 is a mean-squared angular amplitude of the motion and τ_c is the correlation time lying in the sub- and nanosecond time ranges. In this work, the ESE technique was applied to spin-labeled amphiphilic molecules of three different kinds embedded in bilayers of fully saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and mono-unsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipids. Two-pulse ESE data revealed the appearance of stochastic librations above 130 K, with the parameter 〈α²〉τ_c obeying the Arrhenius type of temperature dependence and increasing remarkably above 170–180 K. A comparison with a dry sample suggests that onset of motions is not related with lipid internal motions. Three-pulse ESE experiments (resulting in stimulated echos) in DPPC bilayers showed the appearance of slow molecular rotations above 170–180 K. For D₂O-hydrated bilayers, ESE envelope modulation experiments indicate that isotropic water molecular motions in the nearest hydration shell of the bilayer appear with a rate of ~?10⁵ s^?1 in the narrow temperature range between 175 and 179 K. The similarity of the experimental data found for three different spin-labeled compounds suggests a cooperative character for the ESE-detected molecular motions. The data were interpreted within a model suggesting that dynamical transition is related with overcoming barriers, of 10–20 kJ/mol height, existing in the system for the molecular reorientations.     

Modulation of Fluorophore Environment in Host Membranes of Varying Charge

The net electrical charge of the biological membrane represents an important parameter in the organization, dynamics and function of the membrane. In this paper, we have characterized the change in the microenvironment experienced by a membrane-bound fluorescent probe when the charge of the phospholipids constituting the host membrane is changed from zwitterionic to cationic with minimal change in the chemical structure of the host lipid. In particular, we have explored the difference in the microenvironment experienced by the fluorescent probe 2-(9-anthroyloxy)stearic acid (2-AS) in model membranes of zwitterionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cationic 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (EPOPC) which are otherwise chemically similar, using the wavelength-selective fluorescence approach and other fluorescence parameters. Our results show that the microenvironment experienced by a membrane probe such as 2-AS is different in POPC and EPOPC membranes, as reported by red edge excitation shift (REES) and other fluorescence parameters. The difference in environment encountered by the probe in the two cases could possibly be due to variation in hydration in the two membranes owing to different charges.     

Photoinduced flavin-tryptophan electron transfer across vesicle membranes generates magnetic field sensitive radical pairs

ABSTRACT

Due to the photobiology of the flavoproteins DNA photolyase and cryptochrome, electron transfer reactions between flavins and tryptophan are of significant biological relevance. In addition, electron transfer across vesicle membranes has also seen much attention. In this work, we study the electron transfer reaction between flavins and tryptophan across lipid bilayer membranes in 1,2-dipalmitoyl-sn-glycero-3-phosphocholine small unilamellar vesicles using time-resolved optical absorption microspectroscopy and magnetically affected reaction yield spectroscopy. We demonstrate that riboflavin tetrabutyrate is embedded in the vesicle bilayer and can undergo electron transfer with tryptophan molecules in either the inner water pool or the bulk solution. Remarkably, flavin mononucleotide encapsulated in the inner water pool can undergo electron transfer across the vesicle bilayer to generate a magnetically sensitive radical pair with tryptophan molecules located in the bulk solution. The observed kinetics suggest that back electron transfer occurs between radical pairs generated by diffusive reencounter, either in the vesicle surface water or via electron hopping through degenerate electron exchange.     

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.     

Transfer of pyrene-dietherphosphatidylcholine to serum lipoproteins

The nonhydrolyzable fluorescent diether analog of phosphatidylcholine, 1-O-hexadecyl-2-0-pyrenedecyl-sn-glycero-3-phosphocholine, has been synthesized as a stable probe for the determination of phospholipid transfer to different lipoprotein classes with potential phospholipase activities. After incubation of total human serum with the new probe at 37°C for 3 hours a characteristic partition equilibrium between the main lipoprotein fractions was observed. The fluorescent lipid was not degraded under these conditions and, therefore, served as a marker for choline glycerophospholipid distribution between and transport to serum lipoproteins.     

Influence of Cholesterol on Molecular Motions in Spin-Labeled Lipid Bilayers Observed by Stimulated ESE

Electron spin echo (ESE) study was performed for spin-labeled lipids 1-palmitoyl-2-stearoyl-(5-d)-sn-glycero-3-phosphocholine in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine phospholipid bilayer. Recently (Isaev and Dzuba in J Phys Chem B 112:13285–13291, 2008), three-pulse stimulated ESE was shown to be sensitive to two types of orientational motion of spin labels in phospholipid bilayers at low temperatures (~100–150 K). The first one is fast stochastic libration, with correlation time on the nanosecond time scale. The second one is slow rotational motion, developing on the accessible for measurements microsecond time scale in a small range of reorientation angles, ~0.1°–1°. These two types of motions may be easily discriminated by dependences of the echo decay rates on the time delays between the pulses. The presence of cholesterol in lipid bilayers is found to suppress remarkably rotational motions, while on the contrary stochastic librations seem to become somewhat enhanced. These results evidence that cholesterol increases the long-time stability of lipid orientations in the bilayer, with simultaneous increase of fast fluctuations of these orientations. The former may be related to the known condensing effect of cholesterol and to raft formations, while the latter to the ordering effect.     

Tracing Lysozyme-Lipid Interactions with Long-Wavelength Squaraine Dyes

The applicability of the two newly commercial available squaraine labels Square-670-NHS and Seta-635-NHS to exploring protein-lipid interactions has been evaluated. The labels were conjugated to lysozyme (Lz) (squaraine-lysozyme conjugates below referred to as Square-670-Lz and Seta-635-Lz), a structurally well-characterized small globular protein displaying the ability to interact both, electrostatically and hydrophobically with lipids. The lipid component of the model systems was represented by lipid vesicles composed of zwitterionic lipids egg yolk phosphatidylcholine (PC) and 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), and their mixtures with anionic lipids either beef heart cardiolipin (CL) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), respectively. Fluorescence intensity of Square-670-Lz was found to decrease upon association with lipid bilayer, while the fluorescence intensity of Seta-635-Lz displayed more complex behavior depending on lipid-to-protein molar ratio. Covalent coupling of squaraine labels to lysozyme exerts different influence on the properties of dye-protein conjugate. It was suggested that Square-670-NHS covalent attachment to Lz molecule enhances protein propensity for self-association, while squaraine label Seta-635-NHS is sensitive to different modes of lysozyme-lipid interactions—within the L:P range 6–11, when hydrophobic protein-lipid interactions are predominant, an aggregation of membrane-bound protein molecules takes place, thereby decreasing the fluorescence intensity of Seta-635-Lz. At higher L:P values (from 22 to 148) when electrostatic interactions are enhanced fluorescence intensity of Seta-635-Lz increases with increasing lipid concentrations.     

Liposome destruction by hydrodynamic cavitation in comparison to chemical,physical and mechanical treatments

Liposomes are widely applied in research, diagnostics, medicine and in industry. In this study we show for the first time the effect of hydrodynamic cavitation on liposome stability and compare it to the effect of well described chemical, physical and mechanical treatments. Fluorescein loaded giant 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) lipid vesicles were treated with hydrodynamic cavitation as promising method in inactivation of biological samples. Hydrodynamic treatment was compared to various chemical, physical and mechanical stressors such as ionic strength and osmolarity agents (glucose, Na⁺, Ca²⁺, and Fe³⁺), free radicals, shear stresses (pipetting, vortex mixing, rotational shear stress), high pressure, electroporation, centrifugation, surface active agents (Triton X-100, ethanol), microwave irradiation, heating, freezing-thawing, ultrasound (ultrasonic bath, sonotrode). The fluorescence intensity of individual fluorescein loaded lipid vesicles was measured with confocal laser microscopy. The distribution of lipid vesicle size, vesicle fluorescence intensity, and the number of fluorescein loaded vesicles was determined before and after treatment with different stressors. The different environmental stressors were ranked in order of their relative effect on liposome fluorescein release. Of all tested chemical, physical and mechanical treatments for stability of lipid vesicles, the most detrimental effect on vesicles stability had hydrodynamic cavitation, vortex mixing with glass beads and ultrasound. Here we showed, for the first time that hydrodynamic cavitation was among the most effective physico-chemical treatments in destroying lipid vesicles. This work provides a benchmark for lipid vesicle robustness to a variety of different physico-chemical and mechanical parameters important in lipid vesicle preparation and application.     

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.     

Structure and interaction potentials in solid-supported lipid membranes studied by X-ray reflectivity at varied osmotic pressure

Highly oriented solid-supported lipid membranes in stacks of controlled number N ≃ 16 (oligo-membranes) have been prepared by spin-coating using the uncharged lipid model system 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The samples have been immersed in aqueous polymer solutions for control of osmotic pressure and have been studied by X-ray reflectivity. The bilayer structure and fluctuations have been determined by modelling the data over the full q-range. Thermal fluctuations are described using the continuous smectic Hamiltonian with the appropriate boundary conditions at the substrate and at the free surface of the stack. The resulting fluctuation amplitudes and the pressure-distance relation are discussed in view of the inter-bilayer potential.     

Low-Temperature Molecular Motions in Phospholipid Bilayers in Presence of Glycerol as Studied by Spin-Echo EPR of Spin Labels

Glycerol is used as a cryoprotective agent to protect biological systems under freezing conditions. Electron spin echo (ESE) spectroscopy, a pulsed version of EPR, is capable of studying low-temperature molecular motions of nitroxide spin labels. ESE technique was applied to study molecular motions in phospholipid bilayers prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with added spin-labeled lipids 1-palmitoyl-2-stearoyl-(n-DOXYL)-sn-glycero-3-phosphocholine (n-PCSL, n was optionally 5 or 16). Bilayers were hydrated (solvated) either in pure water or in a 1:1 v/v water–glycerol mixture. In the used ESE approach, there were studied stochastic (or diffusive) orientational vibrations of the molecule as a whole (i.e., stochastic molecular librations). The anisotropic contribution to the echo decay rate, W _anis, was measured, which is proportional, according to theory, to the product of the mean-squared angular amplitude \(\langle \alpha^{ 2} \rangle\) and the correlation time τ _c. W _anis was found to be small below and to sharply increase above 200 K, for the both types of solvents and the both label positions. As compared with hydration by pure water, in presence of glycerol W _anis was larger for the 5th label position while for the 16th one it did not change. Also, for the 5th label position W _anis values were found to be nearly the same as those for a polar spin probe 3,4-dicarboxy-PROXYL which was separately added to the bilayer as a reference and which is assumed to be partitioned only into the solvating shell. These results indicate that motions at the surface of bilayer are governed by the motion of solvating shell while motions in the bilayer interior occur independently. The relation of the obtained data with the dynamical transition phenomenon that is known for biological substances near 200 K from neutron scattering and Mössbauer absorption is discussed.     

Visualization of lipid-receptor interactions on single cells by time-resolved imaging fluorescence microscopy

The physical interaction between plasma-membrane lipids and the epidermal growth factor (EGF)-receptor was investigated on single A431 human epidermoid carcinoma cells by monitoring fluorescence resonance energy transfer (FRET) between exogeneously added fluorescein-EGF (donor) and 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (Bodipy-PC, acceptor) using donor-photobleaching FRET-microscopy. The measured mean FRET-efficiency of 13% is indicative of such a physical interaction and exemplifies the great potential and sensitivity of time-resolved imaging fluorescence microscopy techniques for the study of lipid-receptor interactions on single cells.     

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.