Effects of temperature and pressure on the lateral organization of model membranes with functionally reconstituted multidrug transporter LmrA

To contribute to the understanding of membrane protein function upon application of pressure, we investigated the influence of hydrostatic pressure on the conformational order and phase behavior of the multidrug transporter LmrA in biomembrane systems. To this end, the membrane protein was reconstituted into various lipid bilayer systems of different chain length, conformation, phase state and heterogeneity, including raft model mixtures as well as some natural lipid extracts. In the first step, we determined the temperature stability of the protein itself and verified its reconstitution into the lipid bilayer systems using CD spectroscopic and AFM measurements, respectively. Then, to yield information on the temperature and pressure dependent conformation and phase state of the lipid bilayer systems, generalized polarization values by the Laurdan fluorescence technique were determined, which report on the conformation and phase state of the lipid bilayer system. The temperature-dependent measurements were carried out in the temperature range 5-70 °C, and the pressure dependent measurements were performed in the range 1-200 MPa. The data show that the effect of the LmrA reconstitution on the conformation and phase state of the lipid matrix depends on the fluidity and hydrophobic matching conditions of the lipid system. The effect is most pronounced for fluid DMPC and DMPC with low cholesterol levels, but minor for longer-chain fluid phospholipids such as DOPC and model raft mixtures such as DOPC/DPPC/cholesterol. The latter have the additional advantage of using lipid sorting to avoid substantial hydrophobic mismatch. Notably, the most drastic effect was observed for the neutral/glycolipid natural lipid mixture. In this case, the impact of LmrA incorporation on the increase of the conformational order of the lipid membrane was most pronounced. As a consequence, the membrane reaches a mechanical stability which makes it very insensitive to application of pressures as high as 200 MPa. The results are correlated with the functional properties of LmrA in these various lipid environments and upon application of high hydrostatic pressure and are discussed in the context of other work on pressure effects on membrane protein systems.     

Mechanisms of inhibition of (Na,K)-ATPase by hydrostatic pressure studied with fluorescent probes

To investigate the mechanisms by which hydrostatic pressure inhibits (Na,K)-ATPase, we measured enzyme activity, as a function of pressure and temperature, of purified (Na,K)-ATPase from dog kidney and eel electroplax, and we monitored protein conformation, possible subunit interactions, and the fluidity of the membrane with fluorescent probes. The (Na,K)-ATPase and p-nitrophenylphosphatase activities were inhibited reversibly by pressures below 1.5 kilobars (eel enzyme) and 2.5 kilobars (dog kidney enzyme). Above these pressures, the enzymes were inactivated irreversibly. The plots of 1n(activity) versus pressure were curvilinear; this indicates that the reversible inhibition by pressure involves two or more rate-limiting steps. The calculated activation volumes varied with temperature and pressure and were larger for the (Na,K)-ATPase activity compared to the p-nitrophenylphosphatase activity. The fluorescence polarization of three hydrophobic probes decreased with increasing temperature (10-36 degrees C) and increased with increasing pressure (10(-3)-1.5 kilobars), reversibly, without any evidence of a lipid phase transition. Plots of enzyme activity versus fluorescence polarization of the lipid probes showed an inverse relationship; this indicates that enzyme activity was directly related to the fluidity of the membrane as measured by the lipid probes. Pressure had no effect on the fluorescence polarization of two cardiac glycoside probes nor on the efficiency of resonance energy transfer between either donor and acceptor cardiac glycosides specifically bound to the ouabain sites of different alpha-subunits, or tryptophan and the bound cardiac glycoside probe. These results suggest that dissociation of dimeric alpha-subunits is not related to the inhibition by pressure, and that the cardiac glycoside-complexed enzyme conformation is stabilized by pressure. It is concluded that increased pressure decreases the membrane fluidity which hinders conformational transitions associated with rate-limiting steps of the (Na,K)-ATPase reaction. It is proposed that ion-bound or -occluded forms of (Na,K)-ATPase are stabilized by pressure because they occupy a smaller volume.     

High pressure-induced changes of biological membrane. Study on the membrane-bound Na(+)/K(+)-ATPase as a model system.

In order to study the pressure-induced changes of biological membrane, hydrostatic pressures of from 0.1 to 400 MPa were applied to membrane-bound Na(+)/K(+)-ATPase from pig kidney as a model system of protein and lipid membrane. The activity showed at least a three-step change induced by pressures of 0.1-100 MPa, 100-220 MPa, and 220 MPa or higher. At pressures of 100 MPa or lower a decrease in the fluidity of lipid bilayer and a reversible conformational change in transmembrane protein is induced, leading to the functional disorder of membrane-associated ATPase activity. A pressure of 100-220 MPa causes a reversible phase transition in parts of the lipid bilayer from the liquid crystalline to the gel phase and the dissociation of and/or conformational changes in the protein subunits. These changes could cause a separation of the interface between alpha and beta subunits and between protein and the lipid bilayer to create transmembrane tunnels at the interface. Tunnels would be filled with water from the aqueous environment and take up tritiated water. A pressure of 220 MPa or higher irreversibly destroys and fragments the gross membrane structure, due to protein unfolding and interface separation, which is amplified by the increased pressure. These findings provide an explanation for the high pressure-induced membrane-damage to subcellular organelles.     

A bar model for the pump and channel function of the reconstituted Na+,K+-ATPase

Purified Na+,K+-ATPase is treated with trypsin. The altered enzyme is then reconstituted into liposomes and the change in active and passive Na+,K+-fluxes is recorded. Trypsin treatment transforms the slow passive Na+,K+-fluxes into leaks. The leak formation is correlated with the degree of proteolysis and the associated decrease in Na+,K+-ATPase activity. The active Na+,K+-transport capacity decreases in parallel with the passive transport. It is thus proposed that the Na+,K+-ATPase molecule primarily contains unspecific transmembrane tunnels that are rendered ion-selective by transverse bars of specific length (bar model).     

The effects of temperature, pressure and peptide incorporation on ternary model raft mixtures--a Laurdan fluorescence spectroscopy study

Recently, an increasing evidence accumulated for the existence of lipid microdomains, called lipid rafts, in cell membranes, which may play an important role in many important membrane-associated biological processes. Suitable model systems for studying biophysical properties of lipid rafts are lipid vesicles composed of three-component lipid mixtures, such as POPC/SM/cholesterol, which exhibit a rich phase diagram, including raft-like liquid-ordered/liquid-disordered phase coexistence regions. We explored the temperature, pressure and concentration-dependent phase behavior of such canonical model raft mixtures using the Laurdan fluorescence spectroscopic technique. Hydrostatic pressure has not only been used as a physical parameter for studying the stability and energetics of these systems, but also because high pressure is an important feature of certain natural membrane environments. We show that the liquid-disordered/liquid-ordered phase coexistence regions of POPC/SM/cholesterol model raft mixtures extends over a very wide temperature range of about 50 degrees C. Upon pressurization, an overall ordered membrane state is reached at pressures of approximately 1,000 bar at 20 degrees C, and of approximately 2,000 bar at 40 degrees C. Incorporation of 5 mol% gramicidin as a model ion channel slightly increases the overall order parameter profile in the l(o)+l(d) two-phase coexistence region, probably by selectively partitioning into l(d) domains, does not change the overall phase behavior, however. This behavior is in contrast to the effect of the peptide incorporation into simple, one-component phospholipid bilayer systems.     

Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Structural changes in lipid bilayers and biological membranes caused hydrostatic pressure

By use of neutron diffraction, the structural parameters of oriented multilayers of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine with deuteriocarbon chains/cholesterol (molar ratio 70:30), multilamellar lipid vesicles composed of pure lipids and lipid/cholesterol mixtures, and crystalline purple membrane patches from Halobacterium halobium have been measured at pressures up to 2 kbar. Pressurization of the oriented 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/cholesterol multilayers results in an in-plane compression with the mean deuteriocarbon chain spacing of 4.44 A obtained under ambient conditions decreasing by 3-7% at 1.9 kbar. The thickness for this bilayer increases by approximately equal to 1.5 A, but the net bilayer volume decreases and the isothermal compressibility is estimated to be in the range (-0.1 to -0.6) X 10(-4)/bar at 19.0 degrees C. The d spacings for multilamellar vesicles composed of lipids in the liquid crystalline state and lipid/cholesterol mixtures increase linearly as a function of pressure, suggesting that these bilayers are also compressed in the membrane plane. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and 1,2-distearoyl-sn-glycero-3-phosphatidylcholine MLVs in the gel state, the d spacing decreases with pressure. For 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine, the hexagonally packed chains are anisotropically compressed in the bilayer plane, resulting in a pseudohexagonal chain packing at 1.9 kbar. The bilayer compressibility is (-0.4 or -0.5) X 10(-4)/bar depending on whether the chain tilt increases with pressure or terminal methyl groups of apposing lipid monolayers approach each other.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ion-gated channel induced in planar bilayers by incorporation of (Na+,K+)-ATPase

An ion-gated channel was conferred on a planar lipid bilayer membrane upon incorporation of (Na+,K+)-ATPase. The channel exhibited two conductance states. The high conductance state was only observed when an ion gradient was present across the planar membrane. This state corresponded to an enzyme conformation which was ouabain and vanadate sensitive (i.e. conductance was inhibited by these compounds), while the low conductance state showed no sensitivity to either inhibitor. Single channel conductance behavior was observed when minimal amounts of enzyme were incorporated into the planar bilayer. The observed single channel conductance was 270 +/- 14 picosiemens. Similar transport behavior was observed for enzyme purified from ovine kidney using sodium dodecyl sulfate (anionic), eel electroplax using Lubrol-WX (nonionic), and kidney microsomes. In addition, the data strongly suggest that enzyme from the kidney microsomes was asymmetrically incorporated into the planar bilayer.     

消炎痛对猪胃H~ /K~ -ATP酶质子转运功能的抑制

作为猪胃H~ /K~ -ATPase的非竞争性抑制剂,消炎痛明显抑制H~ /K~ -ATPase泡囊的质子转运功能,造成质子泄漏。在0.15 mg/ml蛋白浓度下,4%的消炎痛结合于H~ /K~ -A- TPase泡囊上。它能渗入膜脂相并显著降低膜的流动性,并使H~ /K~ -ATPase内源荧光受到淬灭。从实验结果看来,消炎痛对猪胃H~ /K~ -ATPase质子转运功能的抑制来自对酶蛋白和膜结构影响两个方面,而非仅抑制酶蛋白本身的功能。     

Does protein kinase C require Ca2+ for activation?

Ca2+ requirement for protein kinase C activation is a matter of controversy. In this report we have examined Ca2+ dependency of the reaction in different assay systems and shown that the enzyme response to Ca2+, as well as diacylglycerol, depends upon phospholipid species, protein substrate and lipid conformation (micelles or sonicates). These results emphasize that the enzyme characteristics as defined in reconstituted membrane systems may not have a physiological relevance.     

Pressure effects on sarcoplasmic reticulum

The irreversible effects of pressure (1-2000 atm) upon the enzymatic activity and structure of the Ca2+-ATPase of sarcoplasmic reticulum were investigated. Sarcoplasmic reticulum vesicles suspended in a medium of 0.1 M KCl, 10 mM imidazole, pH 7.0, 5 mM MgCl2, and 0.5 mM EGTA irreversibly lose their Ca2+ transport and Ca2+-stimulated ATPase activities on exposure to pressures of 800-2000 atmospheres. The pressure-induced inactivation of Ca2+-ATPase is accompanied by inhibition of the formation of phosphorylated enzyme intermediate, an increase in the passive Ca2+ permeability of the membrane, and structural changes in the Ca2+-ATPase as shown by disruption of Ca2+-ATPase membrane crystals, increased susceptibility to tryptic digestion, unmasking of SH groups, and loss of the conformational responses to Ca2+ and vanadate. The sensitivity to pressure is influenced by enzyme conformation. Ca2+ or vanadate + EGTA protect the Ca2+-ATPase against pressure-induced inactivation, implying a greater stability of the enzyme in the E1 and E2 states than in the conformational equilibrium that prevails at low [Ca2+] in the absence of vanadate. Protection against pressure inactivation was also observed in the presence of sucrose, glycerol, ethylene glycol and 1 M KCl, suggesting that water density modifying groups significantly affect the stability of Ca2+-ATPase under pressure.     

Enzymatic Reconstitution of Brain Membrane and Membrane Opiate Receptors

A new method using lysophosphatide and acyl-CoA as detergents has been used to solubilize the rat brain opiate receptor. After solubilization, lysophosphatide and acyl-CoA can be almost completely removed by an enzymatic reaction that uses an acyltransferase from rat liver microsomes and reconstitutes the solubilized receptor in membranous vesicles. Morphological studies performed with negative staining and freeze-fracture electron microscopy revealed that the general appearance and intramembrane particle distribution of fracture faces in the reconstituted membrane are similar to those of the native membrane; this indicates that hydrophobic protein components of the original membrane were incorporated during reconstitution. Reconstituted membrane, however, contained higher levels of phosphatidylcholine and lower levels of cholesterol. The activities of the membrane-bound enzymes Na+, K+-ATPase and Ca2+, Mg2+-ATPase in the reconstituted system were 24 and 3%, respectively, those of the native membrane. Although binding of opiate ligands to the reconstituted membrane was stereospecific and saturable, higher concentrations of some of the unlabeled ligands were required to inhibit binding of the radiolabeled ligands. These changes in receptor characteristics are likely due to changes in lipid composition, physical state, and/or distribution of the lipids in the reconstituted membrane bilayer. This conclusion is supported by an increase in the affinity of opiate ligands for reconstituted membrane after adjustment of the latter's lipid composition to match more closely that of the original membrane. This was accomplished by treatment with phospholipid exchange protein to remove the excess phosphatidylcholine and by incorporation of cholesterol into the reconstituted membrane.     

Effect of hydrostatic pressure on water penetration and rotational dynamics in phospholipid-cholesterol bilayers.

The effect of high hydrostatic pressure on the lipid bilayer hydration, the mean order parameter, and rotational dynamics of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) cholesterol vesicles has been studied by time-resolved fluorescence spectroscopy up to 1500 bar. Whereas the degree of hydration in the lipid headgroup and interfacial region was assessed from fluorescence lifetime data using the probe 1-(4-trimethylammonium-phenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH), the corresponding information in the upper acyl chain region was estimated from its effect on the fluorescence lifetime of and 3-(diphenylhexatrienyl)propyl-trimethylammonium (TMAP-DPH). The lifetime data indicate a greater level of interfacial hydration for DPPC bilayers than for POPC bilayers, but there is no marked difference in interchain hydration of the two bilayer systems. The addition of cholesterol at levels from 30 to 50 mol% to DPPC has a greater effect on the increase of hydrophobicity in the interfacial region of the bilayer than the application of hydrostatic pressure of several hundred to 1000 bar. Although the same trend is observed in the corresponding system, POPC/30 mol% cholesterol, the observed effects are markedly less pronounced. Whereas the rotational correlation times of the fluorophores decrease in passing the pressure-induced liquid-crystalline to gel phase transition of DPPC, the wobbling diffusion coefficient remains essentially unchanged. The wobbling diffusion constant of the two fluorophores changes markedly upon incorporation of 30 mol% cholesterol, and increases at higher pressures, also in the case of POPC/30 mol% cholesterol. The observed effects are discussed in terms of changes in the rotational characteristics of the fluorophores and the phase-state of the lipid mixture. The results demonstrate the ability of cholesterol to adjust the structural and dynamic properties of membranes composed of different phospholipid components, and to efficiently regulate the motional freedom and hydrophobicity of membranes, so that they can withstand even drastic changes in environmental conditions, such as high external hydrostatic pressure.     

Behavior of plant plasma membranes under hydrostatic pressure as monitored by fluorescent environment-sensitive probes

We monitored the behavior of plasma membrane (PM) isolated from tobacco cells (BY-2) under hydrostatic pressures up to 3.5 kbar at 30 °C, by steady-state fluorescence spectroscopy using the newly introduced environment-sensitive probe F2N12S and also Laurdan and di-4-ANEPPDHQ. The consequences of sterol depletion by methyl-β-cyclodextrin were also studied. We found that application of hydrostatic pressure led to a marked decrease of hydration as probed by F2N12S and to an increase of the generalized polarization excitation (GPex) of Laurdan. We observed that the hydration effect of sterol depletion was maximal between 1 and 1.5 kbar but was much less important at higher pressures (above 2 kbar) where both parameters reached a plateau value. The presence of a highly dehydrated gel state, insensitive to the sterol content, was thus proposed above 2.5 kbar. However, the F2N12S polarity parameter and the di-4-ANEPPDHQ intensity ratio showed strong effect on sterol depletion, even at very high pressures (2.5-3.5 kbar), and supported the ability of sterols to modify the electrostatic properties of membrane, notably its dipole potential, in a highly dehydrated gel phase. We thus suggested that BY-2 PM undergoes a complex phase behavior in response to the hydrostatic pressure and we also emphasized the role of phytosterols to regulate the effects of high hydrostatic pressure on plant PM.     

Pressure dependence of pyrene excimer fluorescence in human erythrocyte membranes

The intensity of pyrene excimer fluorescence in human erythrocyte membranes and in sonicated dispersions of the membrane lipid (liposomes) was examined as a function of pressure (1–2080 bar) and temperature (5–40°C). Higher pressure or lower temperature decreased the excimer/monomer intensity ratios. A thermotropic transition was detected in both membranes and liposomes by plots of the logarithm of the excimer/monomer intensity ratio versus 1/K. The transition temperature of the membranes was 19–21°C at 1 bar and 28–31°C at 450 bar, a shift with pressure of approx. 20–22 K per kbar. Corresponding transition temperatures of the liposomes were 21°C at 1 bar and 33°C at 450 bar, a shift of approx. 27 K per kbar. The observed pressure dependence of the thermotropic transition temperature is similar to that reported for phospholipid bilayers and greatly exceeds that of protein conformation changes. In concert with the liposome studies the results provide direct evidence for a lipid transition in the erythrocyte membrane.     

Ion-transporting properties and ATPase activity of (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membranes

A purified (Na+ + K+)-ATPase large subunit obtained from microsomes by water-alcohol extraction was incorporated into a bilayer lipid membrane. The protein formed in the membrane conductance channels which were sensitive to ouabain and selective for monovalent cations. ATP activated these channels in the presence of sodium and potassium ions. When sodium ions were eliminated ATP did not change the conductance of the modified membrane whereas p-nitrophenyl phosphate increased it. The (Na+ + K+)-ATPase large subunit incorporated into bilayer lipid membrane possessed an ATPase activity. The presence of a potential on the membrane was a necessary condition for the enzyme incorporated into a bilayer lipid membrane to show high ATPase activity. Increasing the potential above 100 mV resulted in the closing of conductance channels.     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

Studies on the purified Na+,Mg(2+)-ATPase from Acholeplasma laidlawii B membranes: a differential scanning calorimetric study of the protein-phospholipid interactions

The purified Na+,Mg2(+)-ATPase from the Acholeplasma laidlawii B plasma membrane was reconstituted with dimyristoyl phosphatidylcholine and the lipid thermotropic phase behavior of the proteoliposomes formed was investigated by differential scanning calorimetry. The effect of this ATPase on the host lipid phase transition is markedly dependent on the amount of protein incorporated. At low protein/lipid ratios, the presence of increasing quantities of ATPase in the proteoliposomes increases the temperature and enthalpy while decreasing the cooperativity of the dimyristoyl phosphatidylcholine gel to liquid-crystalline phase transition. At higher protein/lipid ratios, the incorporation of increasing amounts of this enzyme does not further alter the temperature and cooperativity of the phospholipid chain-melting transition, but progressively and markedly decreases the transition enthalpy. Plots of lipid phase transition enthalpy versus protein concentration suggest that at the higher protein/lipid ratios each ATPase molecule removes approximately 1000 dimyristoyl phosphatidylcholine molecules from participation in the cooperative gel to liquid-crystalline phase transition of the bulk lipid phase. These results indicate that this integral transmembrane protein interacts in a complex, concentration-dependent manner with its host phospholipid and that such interactions involve both hydrophobic interactions with the lipid bilayer core and electrostatic interactions with the lipid polar head groups at the bilayer surface.     

The reconstitution and activity of the small multidrug transporter EmrE is modulated by non-bilayer lipid composition

The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.     

Destabilization of a lipid non-bilayer phase by high pressure

Pressure is found to destabilize the non-bilayer phase with respect to the bilayer in a model lipid system. The lamellar to inverted hexagonal (H₁₁) phase transition of aqueous egg phosphatidylethanolamine is shifted to higher temperatures by hydrostatic pressure. The slope of the increase in transition temperature is constant to beyond 300 bar, and is greater than that seen for other lipid phase transitions. This behavior is consistent with the hypothesis that increasing chain disorder drives the conversion from the bilayer into the hexagonal phase. If this non-bilayer lipid phase is an intermediate in membrane fusion, then pressure should inhibit the process. This may explain the inhibition of chemical transmission at neural synapses by pressure.