Cold-induced lipid phase transitions

The structural organization of biological membranes is largely determined by the weak interactions existing between their components and between these components and their aqueous environment. These interactions are particularly sensitive to changes in temperature and hydration. The factors influencing membrane lipid phase behaviour are briefly reviewed and used to develop a phase-separation model describing the response of biological membranes to stress. The factors affecting the interaction of cryoprotectants with membrane lipids are explored and their role in the stabilization of membrane organization at low temperatures discussed. It is suggested that the basis of their protective action lies in an ability to preserve the balance of interactions between membrane components at low temperatures at a level similar to that existing under physiological conditions.     

Isothermal lipid phase transitions

In liotropic lipid systems phase transitions can be induced isothermally by changing the solvent concentration or composition; alternatively, lipid composition can be modified by (bio)chemical means. The probability for isothermal phase transitions increases with the decreasing transition entropy; it is proportional to the magnitude of the transition temperature shift caused by transformation-inducing system variation. Manipulations causing large thermodynamic effects, such as lipid (de)hydration, binding of protons or divalent ions and macromolecular adsorption, but also close bilayer approach are, therefore, likely to cause structural lipid change(s) at a constant temperature. Net lipid charges enhance the membrane susceptibility to salt-induced isothermal phase transitions; a large proportion of this effect is due to the bilayer dehydration, however, rather than being a consequence of the decreased Coulombic electrostatic interactions. Membrane propensity for isothermal phase transitions, consequently, always increases with the hydrophilicity of the lipid heads, as well as with the desaturation and shortening of the lipid chains. Upon a phase change at a constant temperature, some of the interfacially bound solutes (e.g. protons or calcium) are released in the solution. Membrane permeability and fusogenicity simultaneously increase. In mixed systems, isothermal phase transitions, moreover, may result in lateral phase separation. All this opens up ways for the involvement of isothermal phase transitions in the regulation of biological processes.     

A theoretical model for lipid monolayer phase transitions

We present a theoretical model for the liquid-expanded to liquid-condensed phase transition observed in many phospholipid monolayer films. The total two-dimensional pressure in the model is the sum of the hydrocarbon chain pressure and the surface pressure. The hydrocarbon chain pressure is calculated in an extended version of a model published earlier. The surface pressure results from a lowering of the surface tension in the monolayer over that of pure water, thus producing a force on a Langmuir float. When these two contributions are added, $\text{π/A}$ isotherms are obtained which have slope discontinuities very similar to those observed experimentally. These results indicate that a successful model for lipid phase behavior must consider the interactions between head groups and water as well as cooperative hydrocarbon chain melting.     

A theoretical model for lipid monolayer phase transitions.

We present a theoretical model for the liquid-expanded to liquid-condensed phase transition observed in many phospholipid monolayer films. The total two-dimensional pressure in the model is the sum of the hydrocarbon chain pressure and the surface pressure. The hydrocarbon chain pressure is calculated in an exteded version of a model published earlier. The surface pressure results from a lowering of the surface tension in the monolayer over that of pure water, thus producing a force on a Langmuir float. When these two contributions are added, pi/A isotherms are obtained which have slope discontinuities very similar to those observed experimentally. The results indicate that a successful model for lipid phase behavior must consider the interactions between head groups and water as well as cooperative hydrocarbon chain melting.     

Stratum corneum lipid phase transitions and water barrier properties

In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.     

A model for phase transitions in lipid bilayers and biological membranes

We wish to present an order-disorder model for the observed phase transitions in lipid bilayers and biological membranes. We show that the model may, under certain circumstances, exhibit two phase transitions, one corresponding to positional disordering of entire lipid molecules, and the other corresponding to orientational disordering in the hydrocarbon chains. We then give results of our numerical analysis of the model and compare them with experimental data. Shortcomings of the model and future directions for analyses of this type are also discussed.     

Effect of delta-lysin on the phase transitions of lipid assemblies

X-ray small-angle diffraction, differential scanning calorimetry (DSC), and temperature scanning densitometry (TSD) were used to study the effect of -lysin on the phase transitions of lipid assemblies from 1,2-0-dixehadecyl-sn-glycero-3-phosphoholine (DHPC). The experiments were carried out in excess of water in a temperature range of 0–55 °C, and at low peptide concentrations between 10^-4 and 10^-2 moles peptide per mole phospholipid. The incorporation of -lysin into lipid assemblies alters the lipid structure without significant changes on the temperatures of phase transition from gel to liquid crystalline phase. The temperature of the main transition was nearly unaffected. A reduction in the transition volume of the lipids with increasing concentrations of -lysin was observed. The minor changes in these parameters were interpreted as long-range structural changes caused by the peptide incorporation. The results are discussed in terms of the concept of cooperative phase transition of entire clusters occurring within a membrane implying that relative stable domains of gel phase, and liquid crystalline phase co-exist.     

Coupled membrane lipid miscibility and phosphotyrosine-driven protein condensation phase transitions

Lipid miscibility phase separation has long been considered to be a central element of cell membrane organization. More recently, protein condensation phase transitions, into three-dimensional droplets or in two-dimensional lattices on membrane surfaces, have emerged as another important organizational principle within cells. Here, we reconstitute the linker for activation of T cells (LAT):growth-factor-receptor-bound protein 2 (Grb2):son of sevenless (SOS) protein condensation on the surface of giant unilamellar vesicles capable of undergoing lipid phase separations. Our results indicate that the assembly of the protein condensate on the membrane surface can drive lipid phase separation. This phase transition occurs isothermally and is governed by tyrosine phosphorylation on LAT. Furthermore, we observe that the induced lipid phase separation drives localization of the SOS substrate, K-Ras, into the LAT:Grb2:SOS protein condensate.     

A self-consistent chain model for the phase transitions in lipid bilayer membranes

We presented a mechanical model of a lipid bilayer membrane. The internal conformations of a polar head group and double hydrocarbon chains in a lipid molecule were described on the basis of the isomeric bond-rotation scheme. The thermodynamic properties of the lipid membranes were represented by a density matrix that described the rotational isomeric states of the head groups and chains. The parameters that determined the density matrix were obtained in the presence of the intermolecular interactions, which depend on the conformation of the molecules. The interchain interaction was given by the Kihara potential, which depends on the shape of the chains. The Coulomb interaction between the polar head groups and the lateral pressure were considered. The calculation was made for the three lipid molecules corresponding to DMPC, DPPC, and DSPC. The model agreed well with the following experimental results: the temperature, the latent heat of the gel-to-liquid crystalline phase transition, the temperature dependencies of (a) the intermolecular distance, (b) the number of gauche bonds in a hydrocarbon chain, (c) the order parameter for the bond orientation, (d) the volume of the membrane, (e) the thermal expansion coefficients, and (f) the birefringence.     

A new method for the demonstration of phase transitions in lipid films

Monolayers of l-α-lecithin (β,γ-dimyristoyl), l-α-lecithin (β,γ-dipalmitoyl) and egg phosphatidylethanolamine were formed on a water surface at a sufficiently low temperature. The rate of evaporation of water was measured as a function ofincreasing temperature.In agreement with previous reports, the monomolecular films had a marked influence on the rate of evaporation of water, and the passage of water molecules through the monolayer was retarded.Using films in which phase transitions can be observed at a specific transition temperature [l-α-lecithin (β,γ-dimyristoyl) at 23°C and l-α-lecithin (β,γ-dipalmitoyl) at 41 °C], an unexpected decrease of water permeability was measured at this particular temperature. The retardation suggests that water transport through the film is hindered. It is thought that these results are best explained by an enhanced interaction between the water molecules and the chains of the lipid molecules above the phase transition temperature.     

Initiation of anion-selective ionic channels in bilayer membranes at lipid phase transitions

A study was carried out to electric parameters of single ionic channels initiated at phase transition of bromidmetilate 1,2-distearoyl-rac-glycero-3-(O-beta-dimethylaminoethyl)-methylphosphonate, whose molecules under conditions given below are possibly charged. It has been shown that changes of transmembrane current appear at phase transition temperature. Comparison between ionic selectivity of channels initiated at Tph.t in the membranes of DSL and its phosphate analog suggests that the channel walls initiated at phospholipid phase transitions are covered with polar groups of molecules.     

A theoretical model for lipid mixtures, phase transitions, and phase diagrams.

We present a new model for the thermodynamic properties of lipid bilayers. The model consists of a system of hard cylinders of varying radii that correspond to the different molecular radii of lipids having different numbers of gauche rotations in their chains. Scaled particle theory is used to provide an accurate estimate of the entropy of packing of the cylinders. To apply the model to bilayers we introduce a semiempirical attractive potential energy. Once the form of this potential is chosen, we adjust one parameter, the interaction strength, so that the model fits the transition temperatures and entropies for various phospholipids. The model then agrees quite well with other published data for these systems. We also directly generalize our model to lipid mixtures, and we obtain phase diagrams that we compare to existing data for these systems. We use the model to describe lipid protein interactions in bilayers as well.     

Theory of lipid bilayer phase transitions as detected by fluorescent probes

Summary We present a quantitative theory that relates the fluorescence intensityvs. temperature (I vs. T) profile of a fluorescent-labeled two-component lipid bilayer to the phase diagram of the bilayer and the partition coefficientK of the fluorophore between fluid and solid phases of the bilayer. We show how the theory can be used to evaluateK from experimentalI vs. T profiles and the appropriate phase diagrams as well as to understand the different shapes ofI vs. T profiles obtained with particular fluorophores and phase diagrams. Using calculatedI vs. T graphs, we discuss the meaning of parameters, such as midpoint of the phase transition and onset and termination of a transition, which are often used to characterize phase transitions on the basis of fluorescence intensityvs. temperature profiles.     

Rupture and transformation of lipid bilayer membranes at thermal phase transitions

Nuclear magnetic resonance was used to study dimyristoylphosphatidylcholine vesicles. Loss of vesicle contents and transformation to more extended bilayer structures near the gel to liquid crystalline phase transition is related to potential cell membrane damage on lowering environmental temperatures.     

Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR

A model membrane system composed of egg sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol was studied with static and magic angle spinning (31)P NMR spectroscopy. This model membrane system is of significant biological relevance since it is known to form lipid rafts. (31)P NMR under magic angle spinning conditions resolves the SM and DOPC headgroup resonances allowing for extraction of the (31)P NMR parameters for the individual lipid components. The isotropic chemical shift, chemical shift anisotropy, and asymmetry parameter can be extracted from the spinning side band manifold of the individual components that form liquid-ordered and liquid-disordered domains. The magnitude of the (31)P chemical shift anisotropy and the line width is used to determine headgroup mobility and monitor the gel-to-gel and gel-to-liquid crystalline phase transitions of SM as a function of temperature in these mixtures. Spin-spin relaxation measurements are in agreement with the line width results, reflecting mobility differences and some heterogeneities. It will be shown that the presence of DOPC and/or cholesterol greatly impacts the headgroup mobility of SM both above and below the liquid crystalline phase transition temperature, whereas DOPC displays only minor variations in these lipid mixtures.     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

X-ray diffraction studies of lipid phase transitions in cholesterol-rich membranes at sub-zero temperatures

In X-ray diffraction studies of hydrated (greater than 60%) cholesterol/dioleoylphosphatidylcholine mixtures the lipid packing band showed an abrupt transition from liquid crystal-type to gel-type position and definition at a temperature which decreased progressively to almost -50 degrees C as the proportion of cholesterol was increased to a saturation level of about 50 mol%. Plots of transition temperature against composition (mol% cholesterol) and of peak position against composition provided evidence of a significant change in phospholipid configuration at about 20 mol% cholesterol. However, the data overall suggested a uniform dispersion of the cholesterol molecules in the phospholipid bilayer at all concentrations up to the saturation point. Parallel studies of hydrated lipid extract of erythrocyte membranes and of several cholesterol-rich membrane preparations showed a similar overall change from liquid crystal-type packing at +20 degrees C to a gel-type packing at -30 degrees C to -40 degrees C but without displaying a defined transition temperature.     

Broad lipid phase transitions in mammalian cell membranes measured by Laurdan fluorescence spectroscopy

Employing fluorescence spectroscopy and the membrane-embedded dye Laurdan we experimentally show that linear changes of cell membrane order in the physiological temperature regime are part of broad order-disorder-phase transitions which extend over a much broader temperature range. Even though these extreme temperatures are usually not object of live science research due to failure of cellular functions, our findings help to understand and predict cell membrane properties under physiological conditions as they explain the underlying physics of a broad order-disorder phase transition. Therefore, we analyzed the membranes of various cell lines, red blood cell ghosts and lipid vesicles by spectral decomposition in a custom-made setup in a temperature range from ?40 °C to +90 °C. While the generalized polarization as a measure for membrane order of artificial lipid membranes like phosphatidylcholine show sharp transitions as known from calorimetry measurements, living cells in a physiological temperature range do only show linear changes. However, extending the temperature range shows the existence of broad transitions and their sensitivity to cholesterol content, pH and anaesthetic. Moreover, adaptation to culture conditions like decreased temperature and morphological changes like detachment of adherent cells or dendrite growth are accompanied by changes in membrane order as well. The observed changes of the generalized polarization are equivalent to temperature changes dT in the range of +12 K < dT < -6 K.