Phospholipid Phase Transition Research Articles

Phospholipid phase transitions as revealed by NMR

Aqueous dispersions of phospholipids can adopt a range of polymorphic phases which include bilayer and non-bilayer forms. Within the bilayer form, laterally separated phases may be induced as a result of surface electrostatic associations, thermotropic behaviour, lipid-protein interactions or because of molecular mismatch between chemically distinct phospholipids. Nuclear magnetic resonance (NMR) methods, designed to exploit the properties of either indigenous nuclei or isotopic labels introduced specifically into a phospholipid, can be used in some cases to describe the molecular properties and behaviour of phospholipids in both macroscopically distinct phases and in molecularly distinct phases within the same polymorphic state. If the molecular motion of phospholipids in co-existing phases is sufficiently different, NMR methods can, in principle, give estimates of the life-time of the phases and the rate of molecular exchange between the phases.

Effects of small organic molecules on phospholidpid phase transitions

Small organic molecules are known to exhibit a wide spectrum of physiological or pharmacological effects and many of them are thought to be membrane associated. Therefore a great number of studies is devoted to the interaction between these molecules and phospholipid model membranes. Results obtained for molecular species of varying hydrophobic/hydrophilic balances will be described. It will be shown that, in general, these different molecules induce similar effects on phospholipid phase transitions, although they are located differently in the membrane. Detailed studies of these interactions will help to understand these processes on a molecular level.

Phospholipid phase transitions in model and biological membranes as studied by infrared spectroscopy

Fourier transform infrared (FT-IR) spectroscopy is an extremely powerful yet non-perturbing physical technique for monitoring the conformation and dynamics of all portions of the phospholipid molecule. In this brief review we summarize some recent FT-IR spectroscopic studies of phospholipid phase transitions in model lipid bilayer and in biological membranes which illustrate the great utility of this technique. We show that FT-IR spectroscopy can accurately monitor the gel to liquid-crystalline phase transition and can provide a large amount of detailed information about phospholipid structure and organization in both the gel and liquid-crystalline states of lipid bilayers.

Phospholipid phase transitions: kinetics and structural mechanisms

A brief review is given on the principles and methods used to investigate structural phase transitions in phospholipid supramolecular structures. The conceptual differences of approaches close to and far from equilibrium are addressed, and the consequences in terms of the limits of interpretation for different types of methods, in particular referring to jump-relaxation and steady-state techniques, are surveyed. With the emphasis on connecting dynamic and structural information, the results obtained so far from different techniques are reviewed, and the open questions addressed. The more recent advances by millisecond time-resolved X-ray diffraction with synchrotron radiation and their main results obtained for transitions triggered by IR-laser temperature jumps are summarized. As a major novel aspect in the field, the necessity of considering martensitic, diffusionless transformation mechanisms and the occurrence of intermediate structures is highlighted.

Polymorphic phospholipid phase transitions as tools to understand peptide-lipid interactions

The effect of peptides on bilayer → non-bilayer phase transitions can be used as a tool to investigate the molecular aspects of peptide-lipid interactions. In this contribution the action on membranes of the peptide antibiotic gramicidin A and the bee venom component melittin are compared. Although the know structures and locations of these peptide upon membrane binding are very different, their actions on membranes show striking parallels. A general model is proposed that explains the seemingly complex peptide-lipid interactions by making use of simple concepts.

General features of phospholipid phase transitions

The phase transitions that take place in hydrated phospholipid dispersions are reviewed in terms of the lyotropic and thermotropic mesomorphism. The thermodynamics of the phase transitions are discussed, including the various contributions to the shifts in phase transition temperatures. Particular attention is given to the phase transitions involving the lamellar or lipid bilayer phase, in view of the relevance to the lipid component of biological membranes. These transitions include especially the chain-melting transition and the transformation to non-lamellar phases.

Theoretical models of phospholipid phase transitions

A brief review is given of the theoretical models and calculational methods used to study the main phase transition in phospholipid membranes with particular emphasis on recent progress. The models are discussed in terms of the mechanical variables and the various interactions which are necessary to describe the phase transition. Theoretical results are presented for the transitional properties and how the phase equilibria are affected by “impurities” such as cholesterol, proteins and anaesthetics.

A dynamical model for phase transitions of lipids induced by calcium ions

Lipid membranes exist essentially in two different phases. A phase transition can be triggered off either by changing the temperature or, isothermally, by varying an external factor such as ionic concentration, pH, organic solvents, etc. Since the isothermal transition may be induced at physiological temperature, it may play an important regulatory role in diverse cellular functions. Based on the Landau-Ginsburg theory, the thermotropic transitions of lipids has been described by a number of models. In the present work a dynamical model for an isothermal phase transition of phospholipids induced by ionic binding is proposed. The properties of the model show that by ionic binding, phospholipids may form spatial heterogeneous distributions of lipids in fluid and crystalline phases. This heterogeneity possibly being the cause of membrane instabilities, which favour enhanced vesicle fusions observed in the presence of Ca2+.

A photophysical model for diphenylhexatriene fluorescence decay in solvents and in phospholipid vesicles

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in pure solvents and in phospholipid vesicles has been measured using frequency domain fluorometry. Data analysis uses a model with two energetically close excited states. The model explains the high quantum yield and the double exponential decay of DPH observed in some pure solvents and in phospholipid vesicles. This model assumes that after excitation to a first excited state, there is a rapid interconversion to a lower excited state and that most of the emission occurs from this state. The interconversion rates between the two excited states determine the average lifetime. For DPH in solvents, we find that the interconversion rates are solvent and temperature dependent. For DPH in phospholipid vesicles, we find that the back reaction rate from excited state 2 to excited state 1 (R12) is what determines the fluorescence properties. The phospholipid phase transition affects only this back reaction rate. The model was analyzed globally for a range of solvents, temperatures and vesicle composition. Of the six parameters of the model, only two, the interconversion rates between the two excited states, varied in all different samples examined. For DPH in phospholipid vesicles, there is an additional feature of the model, which is related to the apparent distribution of the rate R12. Significantly better fits were obtained using a continuous lorentzian distribution of interconversion rates. The resulting lifetime distribution was asymmetric and showed a definite narrowing above the phase transition.

Phase diagrams of pseudo-binary phospholipid systems IV. Preliminary results about the effects of LiCl and CaCl 2 on the phase transitions of amphoteric phospholipids in aqueous dispersions

Differential scanning calorimetry and freeze-fracture electron microscopy were applied to study the effects of LiCl and CaCl 2 on the lyotropic and thermotropic properties of dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine in disperions with excess water. Significant changes of the phase transition temperatures and phase transition enthalpies were observed. The phase diagrams of the systems dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylethanolamine/H 2O/LiCl and dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylethanolamine/H 2O/CaCl 2, showed that the influence of Li +- and Ca 2+-ion does not effect fundamental changes in the miscibility properties of the two amphoteric phospholipids.

Fast time-resolved X-ray diffraction for studying laser T-jump-induced phase transitions

Phase transitions of phospholipid/water systems, triggered by a short 1–2 ms infrared laser heat pulse (Er-laser, wavelength: 1.5 μm, energy 1–2 J), were studied by recording their real time X-ray powder diffraction patterns at a time resolution down to 0.5 ms using synchrotron radiation. Theoretical calculations of the temperature ( T) profile show that the thermal gradient within the heated region of the aqueous sample is less than 2°C and that the influence of temporal heat diffusion can be neglected on the time scale of these experiments, i.e., ⩽ 5 s. With this technique, combining the fast heating source with a fast X-ray detection system, it is possible to study the molecular spatial rearrangements and to evaluate the kinetics of thermotropic phase transitions of phospholipids in the millisecond time range.

Incorporation of spin-labelled fatty acids into bovine brain clathrin coated vesicles

Stearic acids with a nitroxide radical at selected positions have been incorporated in the phospholipid bilayers of clathrin coated vesicles, uncoated vesicles and sonicated liposomes made from the lipids extracted from the uncoated vesicles. The extent of incorporation was found minimum for stearic acids labeled on C-12 and for bilayers of uncoated vesicles. The ESR spectra of the spin-labeled fatty acids incorporated in the bilayers showed a pronounced temperature dependence (without discontinuity) and a decrease in the hyperfine splitting as the nitroxide group was inserted deeper in the hydrophobic core of the membranes. As abrupt phospholipid phase transition or a phase separation could be excluded. The presence of the external proteins (the clathrin coat) on the membranes was not found to noticeably influence the gradient of flexibility of the fatty acid chains of the phospholipids. The influence of the internal proteins embedded in the bilayers was evidenced by a detailed analysis of the ESR spectra of (7,8)SA in terms of two components: one component arising from the labels surrounded exclusively by phospholipids, the other component arising from labels of reduced mobility perturbed by the vicinity of the proteins. These results support the persistence of lipidic domains in the endocytic vesicles despite the accumulation of receptors which follows their formation.

The effects of A23187 on the phospholipid phase transition of large unilammellar vesicles (LUVs) as detected by ultrasound spectroscopy

The effect of the hydrophobic Ca 2+ ionophore, A23187, on the phospholipid dynamics of large unilamellar vesicle (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG) membranes, as a function of A23187 content, was investigated using techniques sensitive to the phospholipid phase transition. The ultrasonic absorption per wavelength, αλ, was determined with a double crystal acoustic interferometer, as a function of tempertaure and frequency for LUVs in the vicinity of their phospholipid phase transition. Differential scanning calorimetry (DSC) and electron spin resonance (ESR) were also employed to probe the thermodynamics and molecular environment of the hydrocarbon side chains. With increasing A23187 content, the phase transition temperature ( T m _of the LUV suspensions remained near 42.0°C, while the amplitude of αλ at the phase transition incresed dramatically. At T m the relaxation frequency, were αλ max occurs, decreased with A23187 content, suggesting that the relaxation rate of the event responsible for the absorption of ultrasound decreased. The ESR studies showed no change in the fluidity of the bilayer with the inclusion of 2 and 5 mol% A23187 in the C-12 region of the bilayer. Therefore, A23187 in LUV membranes slows the structural relaxation of the hydrocarbon side chains of the phospholipid bilayer at the phase transition.

Abscisic acid-induced microheterogeneity in phospholipid vesicle

Changes in the thermal behavior of DMPC (dimyristoyl-L-phosphatidylcholine) and an equimolar mixture of DMPC and DMPE (dimyristoyl-L-phosphatidylethanolamine) induced by the plant hormone abscisic acid (ABA) have been investigated using fluorescent probes. The fluorescence decay of the hydrophobic probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in these vesicles has been measured using frequency-domain fluorometry, and has been analyzed using both models of discrete exponential components and continuous lifetime distributions. In the DMPC vesicles, using the distributional approach, higher center and width values were observed in the presence of abscisic acid (ABA), indicating a decrease in the dielectric constant of the lipid phase that we attribute to a decrease in the water concentration within the bilayer. Moreover, the presence of ABA in the liposomes increased the phospholipid phase transition temperature. The addition of ABA to the DMPC/DMPE mixture strongly increased the microheterogeneity of the system as reported by the FWHM (full-width at half-maximum) of the distributional approach.

Intermediates in membrane fusion and bilayer/nonbilayer phase transitions imaged by time-resolved cryo-transmission electron microscopy

Bilayer-to-nonbilayer phase transitions in phospholipids occur by means of poorly characterized intermediates. Many have proposed that membrane fusion can also occur by formation of these intermediates. Structures for such intermediates were proposed in a recent theory of these transition mechanisms. Using time-resolved cryo-transmission electron Microscopy (TRC-TEM), we have directly visualized the evolution of inverted phase micro-structure in liposomal aggregates. We have identified one of the proposed intermediates, termed an interlamellar attachment (ILA), which has the structure and dimensions predicted by the theory. We show that ILAs are likely to be the structure corresponding to "lipidic particles" observed by freeze-fracture electron microscopy. ILAs appear to assemble the inverted cubic (III) phase by formation of an ILA lattice, as previously proposed. ILAs are also observed to mediate membrane fusion in the same systems, on the same time scale, and under nearly the same conditions in which membrane fusion was observed by fluorescence methods in earlier studies. These earlier studies indicated a linkage between a membrane fusion mechanism and III phase formation. Our micrographs suggest that the same intermediate structure mediates both of those processes.

Interaction of type I collagen fibrils with phospholipid vesicles

Type I collagen fibrils interact with phosphatidylcholine and phosphatidylglycerol vesicles. Fluorescence polarization of (1,6-diphenyl-1,3,5-hexatriene) DPH-labeled vesicles, circular dichroism and differential scanning calorimetry studies have been performed. The protein-lipid interaction produces a decrease of the enthalpy of the phospholipid phase transition. Positive charges of lysine residues of the protein are involved in the interaction as experiments with succinylated collagen show. The kinetic parameters and the extent of the fibrillogenesis of collagen are modified by the phospholipid vesicles.

The effects of 2H 2O on the phase transition of large unilamellar vesicle (LUV) suspensions as detected by ultrasound spectroscopy and electron spin resonance

The importance of water in the molecular dynamics of large unilamellar vesicle (LUV) suspensions, in which increasing portions of the water were replaced by 2H 2O, was investigated. Determinations of the ultrasonic absorption coefficient per wavelength, αλ, were performed as a function of temperature and frequency for LUVs (LUVs: 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine, DPPC, and dipalmitoylphosphatidylglycerol, DPPG) in the vicinity of their phospholipid phase transition, using a double crystal acoustic interferometer. Electron spin resonance (ESR) and differential scanning calorimetry (DSC) were also employed to probe this system. When increasing portions of the aqueous content of the LUV suspensions were replaced by 2H 2O the phase transition temperature increased from 42.0°C to 42.9°C (indicating an increase in the activation energy of the transition), and the amplitude of αλ at the phase transition increased. However, αλ max as a function of frequency at the phase transition did not change with the addition of 2H 2O, indicating that the relaxation time of the event responsible for the absorption of ultrasound was unaffected. The increase in the activation energy of the transition with the addition of 2H 2O suggested that the mobility of phospholipids near the membrane/aqueous interface was changed. Electron spin resonance (ESR) experiments on LUVs with nitroxide spin probes positioned at the membrane/aqueous interface (5-doxyl stearate and CAT 16) showed that LUVs in 2H 2O have a broader splitting, A max, at the membrane/aqueous interface than do LUVs in H 2O. These results suggest that 2H 2O changes the mobility and/or structure of the phospholipids in the region of the membrane/aqueous interface. This difference in A max was not seen for the probe PC-12-doxyl stearate, which resides at the C-12 position of the bilayer.

Lipolysis of phospholipids in model cholesteryl ester rich lipoproteins and related systems: effect of core and surface lipid phase state.

Porcine pancreatic phospholipase A2 (PL A2) was used as a probe to study the structure of phospholipid domains of dimyristoylphosphatidylcholine (DMPC) vesicles +/- 2% cholesteryl oleate (CO), of discoidal structures formed by the interaction of apolipoprotein E (apoE) with these vesicles, and of large CO/DMPC microemulsion particles +/- apoE. Results of phospholipid hydrolysis over a range of temperatures were compared with the thermal transitions of the lipid components of the particles as measured by differential scanning calorimetry. These studies revealed that DMPC vesicles were most susceptible to digestion at or near the transition temperature. A similar result was obtained with DMPC/apoE disks; however, these particles were hydrolyzed over a broader temperature range than the vesicles. DMPC/CO vesicles were resistant to hydrolysis at every temperature tested; however, discoidal structures formed by interaction of apoE with these vesicles were hydrolyzed maximally above their thermal transition. Large microemulsion particles of CO and DMPC were poor substrates for the enzyme at every temperature; binding of apoE to these particles improved the ability of PL A2 to hydrolyze the phospholipid. These results suggest that the curvature of the surface, the presence of dissolved cholesteryl ester, and the binding of protein have profound effects on the temperature and breadth of the phospholipid phase transition and on the accessibility of the phospholipid to hydrolysis by PL A2.

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (αλ), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG)). It has been found that αλ reaches a maximum ( αλ) max at the phase transition temperature ( t m) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca 2+ and Mg 2+), added to LUV suspensions, shifted ( αλ) max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the αλ versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in t m suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, αλ as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles.

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)