Models of haloadaptation in bacterial membranes

Abstract Cell membranes consist of a complex assortment of amphipathic lipids. These lipids exist in one of three phases in aqueous systems at the growth temperature of the organism: namely, lamellar gel, lamellar liquid-crystalline or hexagonal-II. The phase behaviour is modified by interaction of the lipids with other membrane components and electrolytes. A stable membrane structure is achieved when the polar and non-polar interactions are balanced such that a durable bilayer arrangement is formed into which the various membrane proteins are integrated. The effect of surface charge on phase domain behaviour of the membrane lipids and the modulation by electrolytes is crucial to understanding how halophiles adapt to high-salt environments.     

Lipid polymorphism

In this review the polymorphic phase behaviour of several of the major classes of lipids found in biological membranes, both in isolation and also in mixtures, is briefly described. Emphasis is given to the ability of many membrane lipids to adopt non-lamellar phases in response to a variety of factors such as temperature, the presence of divalent cations or changes in pH. The phase behaviour of mixed lipid systems and factors which can modulate the phase preferences of such systems are considered in some detail particularly with regard to the effect of cholesterol upon lipid polymorphism.     

Phase behaviour of the membrane lipids of the thermophilic blue-green alga Anacystis nidulans

The phase behaviour of total membrane lipid extracts of the blue-green alga Anacystis nidulans is compared with that of the individual lipid classes present in such extracts using fluorescence probe, differential scanning calorimetry, wide-angle X-ray diffraction and freeze-fracture techniques. Marked differences are observed in the properties of the isolated lipids as compared to the total lipid extracts. In particular, purified samples of monogalactosyldiacylglycerol and phosphatidylglycerol form complex high melting-point gel phases on storage which are not found in the membrane extracts. Addition of Mg²⁺ ions to the extracts is also shown to lead to an extensive phase separation of monogalactosyldiacylglycerol from the extracts. The enthalpy changes associated with phase separations occurring in the lipid extracts are found to be approx. 30% higher than those for the corresponding membranes, suggesting that the presence of other components, such as membrane proteins, may influence the phase behaviour of the lipids. The significance of these observations is discussed in terms of the factors limiting the stability of membrane systems.     

Thermotropic phase behaviour of the pseudobinary mixtures of DPPC/C12E5 and DMPC/C12E5 determined by differential scanning calorimetry and ultrasonic velocimetry

The present paper reports on the phase behaviour of the pseudobinary aqueous mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/pentaethylene glycol monododecyl ether (C12E5) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine monohydrate (DMPC)/C12E5. Both systems exhibit a variety of mesophases, such as lamellar gel, liquid crystalline and micellar phases. The phase diagrams show peritectic and eutectic behaviours. The existence of a compound complex is established. From the phase diagrams, the temperature dependence of the solubilisation parameters is obtained. The phase diagrams, especially with respect to the solubilisation process were qualitatively explained assuming that the packing of the constituents plays a dominating role. Finally, differential scanning calorimetry and ultrasonic velocimetry are compared concerning their potentials to determine characteristics of phase transitions in pseudobinary phospholipid/surfactant mixtures.     

Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systems) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freeze-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with l-α-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.     

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns.     

Aqueous two-phase polymer partitioning of lipid vesicles of defined size and composition

The kinetics of the partitioning of lipid vesicles containing acidic phospholipids in aqueous two-phase polymer systems are dependent upon the vesicle size; the larger the vesicles, the more readily they absorb to the interfaces between the two polymer phases and hence are cleared from the top phase as phase separation proceeds. The partitioning of neutral lipid vesicles is principally to the bulk interface and is the same in phase systems of both low and high electrostatic potential difference between the two phases (delta psi). The incorporation of negatively charged lipids has two effects upon partition. First, vesicles with negatively charged lipids exhibit increased bottom phase partitioning in phases of low delta psi due to an enhanced wetting of the charged lipids by the lower phase. Second, the presence of a negatively charged group on the vesicle surface results in increased partition to the interface and top phase in phase systems of high delta psi. Differences observed in the partition of vesicles containing various species of negatively charged lipid thus reflect a competition between these two opposing factors.     

Surface respreading after collapse of monolayers containing major lipids of pulmonary surfactant

Isotherms have been obtained near 37 degrees C for a series of repetitive compressions and expansions of monolayers that contain major components of lung surfactant. The minimum surface tension or maximum surface pressure which could be achieved under conditions of dynamic compression, and the rate of return of lipid from excluded phase to the monolayers were measured. Monolayers of pure 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), or of DPPC plus 10 or 30 mol% of the calcium salt of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-rac-glycerol (POPG) (POPG-Ca) achieved very high surface pressures or low surface tensions (near 0 mN m-1), but they showed no return of material from the collapse phases under the test conditions. Monolayers of POPG-Ca alone collapsed at relatively low surface pressures (high surface tensions), but showed good return of material from the collapse phase into the monolayer. Monolayers containing more complex mixtures of lipids (DPPC, phosphatidylglycerol (PG), unsaturated phosphatidylcholine (PC), cholesterol (chol] in ratios similar to those found in surfactant achieved minimum surface tensions intermediate between those of monolayers with less complex compositions. These more complex mixtures showed a better rate of return of lipids from the collapse phases to the monolayer than did simple DPPC-POPG mixtures. 31P-NMR and differential scanning calorimetric investigations of the mixture DPPC/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine(POPC)/POP G/DPPG/chol (10:4:2:1:3) showed that in the bulk phase at 37 degrees C, it was in bilayers in the liquid-crystalline state.     

Protein-induced surface structuring in myelin membrane monolayers

Monolayers prepared from myelin conserve all the compositional complexity of the natural membrane when spread at the air-water interface. They show a complex pressure-dependent surface pattern that, on compression, changes from the coexistence of two liquid phases to a viscous fractal phase embedded in a liquid phase. We dissected the role of major myelin protein components, myelin basic protein (MBP), and Folch-Lees proteolipid protein (PLP) as crucial factors determining the structural dynamics of the interface. By analyzing mixtures of a single protein with the myelin lipids we found that MBP and PLP have different surface pressure-dependent behaviors. MBP stabilizes the segregation of two liquid phases at low pressures and becomes excluded from the film under compression, remaining adjacent to the interface. PLP, on the contrary, organizes a fractal-like pattern at all surface pressures when included in a monolayer of the protein-free myelin lipids but it remains mixed in the MBP-induced liquid phase. The resultant surface topography and dynamics is regulated by combined near to equilibrium and out-of-equilibrium effects. PLP appears to act as a surface skeleton for the whole components whereas MBP couples the structuring to surface pressure-dependent extrusion and adsorption processes.     

Synchrotron X-ray and neutron small-angle scattering of lyotropic lipid mesophases, model biomembranes and proteins in solution at high pressure

In this review we discuss the use of X-ray and neutron diffraction methods for investigating the temperature- and pressure-dependent structure and phase behaviour of lipid and model biomembrane systems. Hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of lipid mesophases, but also because high pressure is an important feature of certain natural membrane environments and because the high pressure phase behaviour of biomolecules is of importance for several biotechnological processes. Using the pressure jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction, the kinetics of different lipid phase transformations was investigated. The techniques can also be applied to the study of other soft matter and biomolecular phase transformations, such as surfactant phase transitions and protein un/refolding reactions. Several examples are given. In particular, we present data on the pressure-induced unfolding and refolding of small proteins, such as Snase. The data are compared with the corresponding results obtained using other trigger mechanisms and are discussed in the light of recent theoretical approaches.     

Temperature-pressure configurational landscape of lipid bilayers and proteins.

High hydrostatic pressure has been used as a physical parameter for studying the stability and energetics of biomolecular systems, such as lipid bilayers and proteins, but also because high pressure is an important feature of certain natural membrane environments. By using a variety of spectroscopic and scattering techniques, the temperature and pressure dependent structure and phase behaviour of various lipid systems and proteins have been studied and are discussed. A thermodynamic approach is presented for studying the stability of proteins as a function of both temperature and pressure. Moreover, the effect of various chaotropic and kosmotropic cosolvents on the temperature- and pressure-dependent structure and stability of proteins is discussed. The results demonstrate that combined temperature-pressure-cosolvent dependent studies can help delineate the free energy landscape of proteins and hence help elucidate which features and thermodynamic parameters are essential in determining the stability of the native conformational state of proteins. We also introduce pressure as a kinetic variable. Applying the pressure-jump relaxation technique in combination with time-resolved synchrotron X-ray diffraction and spectroscopic techniques, the kinetics of un/refolding of lipid mesophases and proteins has been studied. Finally, recent advances in using pressure for studying misfolding and aggregation of proteins will be elucidated.     

A comparison of spin probe ESR, 2H- and 31P-nuclear magnetic resonance for the study of hexagonal phase lipids

We have investigated the feasibility of the various possible magnetic resonance probes of lipids which form non-bilayer phases. As a model system we have used equimolar mixtures of phosphatidylethanolamine (PE) and cholesterol, which exhibit a thermotropic transition from a bilayer to a hexagonal phase. Variable temperature electron spin resonance (ESR) spin probe spectra were obtained using random dispersion and oriented lipid systems. Simultations of the ESR spectra were performed in order to aid in the interpretation of the experimental results for the oriented system. ³¹P- and ²H-nuclear magnetic resonance (NMR) studies were carried out using a deuterated PE. The ESR spin probes in the random dispersions show essentially no effect attributable to the phase transition. However, there are large, reversible effects in the temperature-dependent behaviour for the oriented system. The orientation dependence of the spectra above the transition temperature indicate that the hexagonal phase lipids may spontaneously assume a macroscopic organization on a flat surface. We find, however, that such an organization cannot be unambiguously assigned from the ESR spin probe spectra, and point out a potential difficulty in the interpretation of spin probe spectra in oriented systems. In contrast, the ²H-NMR method provides a reliable monitor of the phase transformation. Taken together, the ²H and ³¹P data indicate that the structure of the headgroup in PE is quite similar in both the bilayer and hexagonal phase. ²H-NMR should be very useful in probing the structural and dynamic characteristics of lipids in non-bilayer phases.     

alpha-methylene ordering of acyl chains differs in glucolipids and phosphatidylglycerol from Acholeplasma laidlawii membranes: (2)H-NMR quadrupole splittings from individual lipids in mixed bilayers

A Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with alpha-deuterated oleic acid. Phosphatidylglycerol (PG), the glucolipids monoglucosyldiacylglycerol (MGlcDAG), diglucosyldiacylglycerol (DGlcDAG) and monoacyldiglucosyldiacylglycerol, and the phosphoglucolipid glycerophosphoryldiglucosyldiacylglycerol (GPDGlcDAG) were purified, and the phase behaviour and molecular ordering for the individual lipids, as well as for mixtures of the lipids, were studied by (2)H-, (31)P-NMR and X-ray scattering methods. The chemical structure of all the A. laidlawii lipids, except PG, has been determined and verified previously; here also the chemical structure of PG was verified, utilising mass spectrometry and (1)H and (13)C high resolution NMR spectroscopy. For the first time, lipid dimers were found in the mass spectrometry measurements. The major findings in this work are: (1) addition of 50 mol% of PG to the non-lamellar-forming lipid MGlcDAG does not significantly alter the transition temperature between lamellar and non-lamellar phases; (2) the (2)H-NMR quadrupole splitting patterns obtained from the lamellar liquid crystalline phase are markedly different for PG on one hand, and DGlcDAG and GPDGlcDAG on the other hand; and (3) mixtures of PG and DGlcDAG or MGlcDAG give rise to (2)H-NMR spectra consisting of a superposition of splitting patterns of the individual lipids. These remarkable features show that the local ordering of the alpha-carbon of the acyl chains is different for PG than for MGlcDAG and DGlcDAG, and that this difference is preserved when PG is mixed with the glucolipids. The results obtained are interpreted in terms of differences in molecular shape and hydrophilicity of the different polar headgroups.     

Effects of Detergent β-Octylglucoside and Phosphate Salt Solutions on Phase Behavior of Monoolein Mesophases

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.     

Effects of Detergent β-Octylglucoside and Phosphate Salt Solutions on Phase Behavior of Monoolein Mesophases

Using small-angle x-ray scattering (SAXS), we investigated the phase behavior of mesophases of monoolein (MO) mixed with additives commonly used for the crystallization of membrane proteins from lipidic mesophases. In particular, we examined the effect of sodium and potassium phosphate salts and the detergent β-octylglucoside (βOG) over a wide range of compositions relevant for the crystallization of membrane proteins in lipidic mesophases. We studied two types of systems: 1), ternary mixtures of MO with salt solutions above the hydration boundary; and 2), quaternary mixtures of MO with βOG and salt solutions over a wide range of hydration conditions. All quaternary mixtures showed highly regular lyotropic phase behavior with the same sequence of phases (Lα, Ia3d, and Pn3m) as MO/water mixtures at similar temperatures. The effects of additives in quaternary systems agreed qualitatively with those found in ternary mixtures in which only one additive is present. However, quantitative differences in the effects of additives on the lattice parameters of fully hydrated mesophases were found between ternary and quaternary mixtures. We discuss the implications of these findings for mechanistic investigations of membrane protein crystallization in lipidic mesophases and for studies of the suitability of precipitants for mesophase-based crystallization methods.     

Partition of lipids between emulsified oil and micellar phases of glyceride-bile salt dispersions

The composition of the emulsified oil and of the micellar phases obtained when a glyceride-fatty acid mixture is dispersed in bile salt solution has been defined. The micellar phase in equilibrium with the emulsified oil phase was obtained by filtration through Millipore filters. The behavior of different lipids in such systems was defined as the partition ratio, micellar/emulsified oil phase (m/o). Partition of fatty acids was found to be strongly dependent on the chain length of the fatty acid and the pH of the dispersion. The curve for partition against pH for oleic acid was interpreted to show a pK(a) for oleic acid in bile salt solution of approximately 7. The partition between micellar and oil phases is given for a series of lipids of different polarity. No significant difference in behavior was found for cholesterol and sitosterol. A relationship was found between the partition m/o and filtration rates through a Millipore filter in micellar solution. The lower the partition coefficient the lower was the rate of filtration. The results obtained are discussed in relation to the mechanism of absorption of fat from the small intestine.     

Surface charge markedly attenuates the nonlamellar phase-forming propensities of lipid bilayer membranes: calorimetric and (31)P-nuclear magnetic resonance studies of mixtures of cationic, anionic, and zwitterionic lipids

The lamellar/nonlamellar phase preferences of lipid model membranes composed of mixtures of several cationic lipids with various zwitterionic and anionic phospholipids were examined by a combination of differential scanning calorimetry and (31)P NMR spectroscopy. All of the cationic lipids utilized in this study form only lamellar phases in isolation. Mixtures of these cationic lipids with zwitterionic strongly lamellar phase-preferring lipids such as phosphatidylcholine form only the lamellar liquid-crystalline phase even at high temperatures, as expected. Moreover, mixtures of these cationic lipids with strongly nonlamellar phase-preferring zwitterionic lipids such as phosphatidylethanolamine exhibit a markedly reduced propensity to form inverted nonlamellar phases, again as expected. However, when mixed with anionic lipids such as phosphatidylserine, phosphatidylglycerol, cardiolipin, or phosphatidic acid, a marked enhancement of nonlamellar phase-forming propensity occurs, despite the fact both components of the mixture are nominally lamellar phase-preferring. An examination of the lamellar/nonlamellar phase transition temperatures and the nature of the nonlamellar phases formed, as a function of temperature and of the composition of the mixture, indicates that the propensity to form inverted nonlamellar phases is maximal in mixtures where the mean surface charge of the membrane surface approaches neutrality and decreases markedly with increases in the density of positive or negative charge at the membrane surface. Moreover, the onset temperatures of the reversed hexagonal phase rise more steeply than do those of the inverted cubic phase as the ratio of cationic and anionic lipids is varied, suggesting that the formation of inverted hexagonal phases is more sensitive to this surface charge effect. These results indicate that surface charge per se is a significant and effective modulator of the lamellar/nonlamellar phase preferences of membrane lipids and that charged group interactions at membrane surfaces may have a major role in regulating this particular membrane property.     

An electron microscopy study of the L2-phase (microemulsion) in a ternary system: Triglyceride/monoglyceride/water

Polar lipids such as monoglycerides are able to form an inverse micellar solution, an L2-phase, of water aggregates in triglyceride oil. This thermodynamically stable liquid phase also fulfils the criteria used to identify microemulsions. The structure of this phase at various compositions has been studied by freeze-fracture electron microscopy. The freeze-fracture images show differently oriented stacks of small smooth lamellae. These observations are consistent with X-ray studies indicating curved lipid bilayers arranged in parallel, separating similarly curved aqueous layers of finite size, forming a higly dynamic structure of ‘medium-range’ order. Studies from different compositions in the ternary systems over the L2-phase in binary monoglyceride/water systems to the pure monoglyceride in the liquid state indicate the occurrence of the same type of lamellar structure, which is proposed to be characteristic for polar lipids forming liquid-crystalline phases in contrast to less polar lipids exhibiting an amorphous structure in the liquid state.     

Measurement and interpretation of fluorescence polarisations in phospholipid dispersions.

An instrument that measures the temperature dependence of fluorescence polarisation and intensity directly and continuously is described. The behaviour of four fluorescent probes bound to a number of well characterised model systems was then examined. The motional properties of the probes were determined from the polarisation and intensity data and were found to be sensitive to the crystalline-liquid crystalline phase transitions in phospholipid vesicles of dimyristoly and dipalmitoly phosphatidylcholine. Binary mixture of dilauroyl and dipalmitoyl phosphatidylcholine show lateral phase separation and in this system the probes parition preferentially into the more 'fluid' phase. In systems that have been reported to contain 'short range order' or 'liquid clustering', such as dioleoyl phosphatidylcholine and liquid paraffin, the motion of the probes was found to have anomalous Arrhenius behaviour consistent with the idea that homogeneous phases were not being sampled. The significance of these findings for the interpretation of the behaviour of fluorescent probes bound to natural membranes is discussed.