Spontaneous Curvature,Differential Stress,and Bending Modulus of Asymmetric Lipid Membranes

Lipid bilayers can exhibit asymmetric states, in which the physical characteristics of one leaflet differ from those of the other. This most visibly manifests in a different lipid composition, but it can also involve opposing lateral stresses in each leaflet that combine to an overall vanishing membrane tension. Here, we use theoretical modeling and coarse-grained simulation to explore the interplay between a compositional asymmetry and a nonvanishing differential stress. Minimizing the total elastic energy leads to a preferred spontaneous curvature that balances torques due to both bending moments and differential stress, with sometimes unexpected consequences. For instance, asymmetric flat bilayers, whose specific areas in each leaflet are matched to those of corresponding tensionless symmetric flat membranes, still exhibit a residual differential stress because the conditions of vanishing area strain and vanishing bending moment differ. We also measure the curvature rigidity of asymmetric bilayers and find that a sufficiently strong differential stress, but not compositional asymmetry alone, can increase the bending modulus. The likely cause is a stiffening of the compressed leaflet, which appears to be related to its gel transition but not identical with it. We finally show that the impact of cholesterol on differential stress depends on the relative strength of elastic and thermodynamic driving forces: if cholesterol solvates equally well in both leaflets, it will redistribute to cancel both leaflet tensions almost completely, but if its partitioning free energy prefers one leaflet over the other, the resulting distribution bias may even create differential stress. Because cells keep most of their lipid bilayers in an asymmetric nonequilibrium steady state, our findings suggest that biomembranes are elastically more complex than previously thought: besides a spontaneous curvature, they might also exhibit significant differential stress, which could strongly affect their curvature energetics.     

Miscibility of phosphatidylcholine binary mixtures in unilamellar vesicles: Phase equilibria

Summary Miscibility among phospholipids with different lipid chain-lengths or with different head groups has attracted a number of research efforts because of its significance in biological membrane structure and function. The general consensus about the miscibility of phosphatidylcholines with varying lipid chainlengths appears to be that binary mixtures of phospholipids with a difference of two carbon atoms in the lipid chain mix well at the main phase transition. Miscibility between phosphatidylcholines with differences of four carbon atoms appears to be inconclusive. Previous reports on the phase transition of binary phospholipid mixtures are concerned mainly with multilamellar vesicles and are mostly limited to the main transition. In the present study, unilamellar vesicles were used and miscibility in binary systems between dimyristoyl-, dipalmitoyl- and distearoyl-phosphatidylcholines at pretransition, as well as main transition temperatures was evaluated by constructing phase diagrams. Two methods were used to monitor the phase transitions: differential scanning microcalorimetry and optical absorbance methods. The optical method has the advantage that unilamellar vesicles of dilute phospholipid concentrations can be used. The liquidus and solidus phase boundaries were determined by the onset temperature of heating and cooling scans, respectively, because the completion temperature of a phase transition has no meaning in binary solutions. Dimyristoyl- and distearoyl-phosphatidylcholines. where the difference in the, lipid chain-length is four carbon atoms, mixed well even at pretransition temperature.     

Advantages and limitations of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3- sn-phosphatidylcholine as a fluorescent membrane probe

We have investigated the behavior of 1-palmitoyl-2-[[2-[4- (6-phenyl-trans-1,3,5-hexatrienyl)phenyl]ethyl]carbonyl]-3-sn -phosphatidylcholine (DPHpPC) in synthetic, multilamellar phosphatidylcholine vesicles. This fluorescent phospholipid has photophysical properties similar to its parent fluorophore, diphenylhexatriene (DPH). DPHpPC preferentially partitioned into fluid phase lipid (Kf/s = 3.3) and reported a lower phase transition temperature as detected by fluorescence anisotropy than that observed by differential scanning calorimetry. Calorimetric measurements of the bilayer phase transition in samples having different phospholipid to probe ratios demonstrated very slight changes in membrane phase transition temperature (0.1-0.2 degree C) and showed no measurable change in transition width. Nonetheless, measurements of probe fluorescence properties suggested that DPHpPC disrupts its local environment in the membrane and may even induce perturbed probe-rich local domains below the phospholipid phase transition. Temperature profiles of steady-state fluorescence anisotropy, limiting anisotropy, differential tangent, and rotational rate were similar to those of DPH below the main lipid phase transition but indicated more restricted rotational motion above the lipid phase transition temperature. As for DPH, the fluorescence decay of DPHpPC could be described by either a single or double exponential both above and below the DPPC phase transition. The choice seemed dependent on the treatment of the sample. The intensity-weighted average lifetime of DPHpPC was roughly 1.5 ns shorter than that of DPH. In summary, the measured properties of DPHpPC and its lipid-like structure make it a powerful probe of membrane structure and dynamics.     

Perturbation of phospholipid membranes by juvenile hormone

The effects of juvenile hormone and its analogs Altozar 4E and ZR-777 5E on the phase properties of liposomes prepared from dipalmitoyl phosphatidyl-choline (DPPC) have been examined by differential scanning calorimetry. Each of these compounds reduced the co-operativity of the gel to liquid-crystalline phase transition, which is manifest as a distinct broadening of the main transition endotherm, and split the transition into two distinguishable components centered at 34 and 37°C. However, there was no significant change in enthalpy of the main phase transition, suggesting that juvenile hormone and its analogs perturb the bilayer primarily in the vicinity of the phospholipid headgroups. Moreover, this perturbation does not appear to influence bilayer permeability since the osmotic swelling rates of liposomes prepared from either phosphatidylcholine or dipalmitoyl phosphatidylcholine that contained up to 33 mol% juvenile hormone were not significantly different from the swelling rates of corresponding liposomes containing no juvenile hormone. Splitting of the transition endotherm into two peaks appeared to be peculiar to compounds possessing juvenile hormone activity. A mixture of fatty acid methyl esters broadened the main transition of DPPC, but did not split the endotherm or shift the transition midpoint, and the insect hormone ecdysone had no discernible effect on the DPPC transition apart from eliminating the pretransition. The data indicate that juvenile hormone and its analogs can modulate the physical properties of phospholipid bilayers, and raise the prospect that some of the physiological effects of this hormone may be achieved through its influence on the molecular organization of membrane lipid.     

A solid-solution theory of anesthetic interaction with lipid membranes: temperature span of the main phase transition

Anesthetics (or any other small additives) depress the temperature of the main phase transition of phospholipid bilayers. Certain anesthetics widen the temperature span of the transition, whereas others do not. The widening in a first-order phase transition is intriguing. In this report, the effects of additive molecules on the temperature and its span were explained by the solid-solution theory. By assuming coexistence of the liquid-crystal and solid-gel phases of lipid membranes at phase transition, the phase boundary is determined from the distribution of anesthetic molecules between the liquid-crystal membrane versus water and between the solid-gel membrane versus water. The theory shows that when the lipid concentration is large or when the lipid solubility of the drug is large, the width of the transition temperature increases, and vice versa. Highly lipid-soluble molecules, such as long-chain alkanols and volatile anesthetics, increase the width of the transition temperature when the lipid:water ratio is large, whereas highly water-soluble molecules, such as methanol and ethanol, do not. The aqueous phase serves as the reservoir for anesthetics. Depletion of the additive molecules from the aqueous phase is the cause of the widening. When the reservoir capacity is large, the temperature width does not increase. The theory also predicts asymmetry of the specific heat profile at the transition.     

Influence of the local anesthetic tetracaine on the phase behavior and the thermodynamic properties of phospholipid bilayers.

We investigated the influence of the local anesthetic tetracaine on the thermodynamic properties and the temperature- and pressure-dependent phase behavior of the model biomembrane 1,2-dimyristoyl-sn-glycero-3-phosphocholine by using volumetric measurements at temperatures ranging from 0 degrees to 40 degrees C and at pressures from ambient up to 1000 bar. The pVT measurements were complemented by temperature-dependent differential scanning calorimetric measurements. Information about the influence of different concentrations of the local anesthetic on the thermodynamic changes accompanying the lipid phase transitions, and on the thermal expansion coefficient, the isothermal compressibility, and the volume fluctuations of the lipids in their different phases, could be obtained from these experiments. The incorporation of tetracaine leads to an overall disordering of the membrane, as can be inferred from the depression of the main transition temperature and the reduction of the volume change at the main lipid phase transition. The expansion coefficient alpha p and the isothermal compressibility chi T of the lipid bilayer are enhanced by the addition of tetracaine and strongly enhanced values of alpha p and chi T, and the lipid volume fluctuations are found in the direct neighborhood of the main phase transition region. As tetracaine can be viewed as a model system for amphiphilic molecules, these results also provide insight into the general understanding of the physicochemical action of amphiphilic molecules on membranes. The experimental results are compared with recent theoretical predictions for the phase behavior of anesthetic-lipid systems, and the biological relevance of this study is discussed.     

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens

Geobacter sulfurreducens is a delta-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.     

Antisymmetry,directional asymmetry,and dynamic morphogenesis

Fluctuating asymmetry is the most commonly used measure of developmental instability. Some authors have claimed that antisymmetry and directional asymmetry may have a significant genetic basis, thereby rendering these forms of asymmetry useless for studies of developmental instability. Using a modified Rashevsky-Turing reaction-diffusion model of morphogenesis, we show that both antisymmetry and directional asymmetry can arise from symmetry-breaking phase transitions. Concentrations of morphogen on right and left sides can be induced to undergo transitions from phase-locked periodicity, to phase-lagged periodicity, to chaos, by simply changing the levels of feedback and inhibition in the model. The chaotic attractor has two basins of attraction-right sidedominance and left side dominance. With minor disturbance, a developmental trajectory settles into one basin or the other. With increasing disturbance, the trajectory can jump from basin to basin. The changes that lead to phase transitions and chaos are those expected to occur with either genetic change or stress. If we assume that the morphogen influences the behavior of cell populations, then a transition from phase-locked periodicity to chaos in the morphogen produces a corresponding transition from fluctuating asymmetry to antisymmetry in both morphogen concentrations and cell populations. Directional asymmetry is easily modeled by introducing a bias in the conditions of the simulation. We discuss the implications of this model for researchers using fluctuating asymmetry as an indicator of stress.     

The planar distributions of surface proteins and intramembrane particles in Acholeplasma laidlawii are differentially affected by the physical state of membrane lipids

We have studied the influence of changes in lipid organization on the planar distribution of two classes of membrane proteins: integral proteins which have amino groups exposed to labelling at the membrane surface by the biotin-avidin-ferritin procedure, and those proteins which penetrate the lipid bilayer sufficiently to be seen as intramembranous particles by freeze-fracture electron-microscopy.When the membranes are examined at temperatures below the lipid phase transition, the first class is dispersed and the second patched. At temperatures in the middle of the transition range, both classes are patched. At temperatures just above the phase transition the first class is dispersed and the second patched, and at temperatures well above the transition both classes are dispersed. Freeze-etch studies of avidin-ferritin-labeled membranes confirmed that the distribution seen by the labeling and the freeze-fracture techniques coexist in single membranes. Thus, there exist two distinct classes of membrane proteins with differential organizational responses to the lipid state.     

Electrical capacitance of lipid bilayer membranes of hydrogenated egg lecithin at the temperature phase transition

Electrical capacitance of the planar bilayer lipid membrane (BLM) formed from hydrogenated egg lecithin (HEL) has been studied during many passages through the phase transition temperature. In contrast to the BLM from individual synthetic phospholipids, membranes from HEL did not demonstrate any capacitance change at the phase transition temperature maximum, as measured by differential scanning calorimeter at 52 degrees C. Instead, two temperatures have been discerned by capacitance records: thickening at 42-43 degrees C and thinning at 57-59 degrees C. The first temperature region is close to the transition temperature of dipalmitoyllecithin, whereas the second is close to that of distearoyllecithin, two main components of the HEL. It was suggested that capacitance measurements were able to reveal a phase separation in the BLM from HEL which was not detected by differential scanning calorimetry. The phase transition of the BLM from the liquid crystal state to the gel state is followed by thickening of the bilayer structure, partly due to a gauche- trans transition of lipid molecules but mainly due to redistribution of the solvent n-decane.     

Roles of membrane structure and phase transition on the hyperosmotic stress survival of Geobacter sulfurreducens

Geobacter sulfurreducens is a δ-proteobacterium bacteria that has biotechnological applications in bioremediation and as biofuel cells. Development of these applications requires stabilization and preservation of the bacteria in thin porous coatings on electrode surfaces and in flow-through bioreactors. During the manufacturing of these coatings the bacteria are exposed to hyperosmotic stresses due to dehydration and the presence of carbohydrates in the medium. In this study we focused on quantifying the response of G. sulfurreducens to hyperosmotic shock and slow dehydration to understand the hyperosmotic damage mechanisms and to develop the methodology to maximize the survival of the bacteria. We employed FTIR spectroscopy to determine the changes in the structure and the phase transition behavior of the cell membrane. Hyperosmotic shock resulted in greatly decreased membrane lipid order in the gel phase and a less cooperative membrane phase transition. On the other hand, slow dehydration resulted in increased membrane phase transition temperature, less cooperative membrane phase transition and a small decrease in the gel phase lipid order. Both hyperosmotic shock and slow dehydration were accompanied by a decrease in viability. However, we identified that in each case the membrane damage mechanism was different. We have also shown that the post-rehydration viability could be maximized if the lyotropic phase change of the cell membrane was eliminated during dehydration. On the other hand, lyotropic phase change during re-hydration did not affect the viability of G. sulfurreducens. This study conclusively shows that the cell membrane is the primary site of injury during hyperosmotic stress, and by detailed analysis of the membrane structure as well as its thermodynamic transitions it is indeed possible to develop methods in a rational fashion to maximize the survival of the bacteria during hyperosmotic stress.     

Effect of free fatty acids on the permeability of 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayer at the main phase transition

We measured the influence of saturated and unsaturated free fatty acids on the permeability and partition of ions into 1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers. The bilayer permeability was measured using the depletion of N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1, 2-dihexadecanoyl-sn-glycero-3-phosphatidylethanolamine (N-NBD-PE) fluorescence as a result of its reduction by dithionite. We observed a distinct increase of dithionite permeability at the main gel-fluid phase transition of DMPC. When vesicles were formed from a mixture of DMPC and oleic acid, the membrane permeability at the phase transition was reduced drastically. Stearic acid and methyl ester of oleic acid have little effect. Similar results in the quenching of pyrene-PC in DMPC vesicles by iodide were obtained. Again, the increase of iodide partition into the lipid phase at the main phase transition of DMPC was abolished by the addition of unsaturated free fatty acids. Free fatty acids, in concentrations up to 5 mol%, do not abolish DMPC phase transition when measured by differential scanning calorimetry. It seems that unsaturated, but not saturated, free fatty acids reduce the lipid bilayer permeability to dithionite and iodide ions at the main phase transition of DMPC, without altering the thermodynamic properties of the bilayer.     

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 relationship between environmental temperature, cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus.

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes. Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might directly be determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Transbilayer coupling mechanism for the formation of lipid asymmetry in biological membranes. Application to the photoreceptor disc membrane.

An equilibrium transmembrane asymmetry in charged lipids is shown to arise as a result of oriented, bipolar proteins in the membrane. The basic interaction giving rise to the asymmetry is between a lipid molecule and a transbilayer potential generated by the asymmetric charge distribution in the protein. Thus, a protein can generate a lipid asymmetry without a direct binding interaction between lipid and protein. The generation of an asymmetry in charged lipid by this mechanism can also lead to a concomitant asymmetry in neutral lipids if deviations from ideality in the lipid mixture are taken into account. It is shown that regular solution theory applied to the lipid phase predicts an asymmetry in all components of a ternary mixture as long as one component is electrostatically oriented according to the mechanism mentioned above. The resulting asymmetry is not strongly salt dependent. The mechanism quantitatively accounts for the experimentally determined phospholipid asymmetry in the rod outer segment disc membrane of the vertebrate photoreceptor.     

The relationship between environmental temperature,cell growth and the fluidity and physical state of the membrane lipids in Bacillus stearothermophilus

A definite and characteristic relationship exists between growth temperature, fatty acid composition and the fluidity and physical state of the membrane lipids in wild type Bacillus stearothermophilus. As the environmental temperature is increased, the proportion of saturated fatty acids found in the membrane lipids is also markedly increased with a concomitant decrease in the proportion of unsaturated and branched chain fatty acids. The temperature range over which the gel to liquid-crystalline membrane lipid phase transition occurs is thereby shifted such that the upper boundary of this transition always lies near (and usually below) the temperature of growth. This organism thus possesses an effective and sensitive homeoviscous adaptation mechanism which maintains a relatively constant degree of membrane lipid fluidity over a wide range of environmental temperatures. A mutant of B. stearothermophilus which has lost the ability to increase the proportion of relatively high melting fatty acids in the membrane lipids, and thereby increase the phase transition temperature in response to increases in environmental temperature, is also unable to grow at higher temperatures. An effective homeoviscous regulatory mechanism thus appears to extend the growth temperature range of the wild type organism and may be an essential feature of adaptation to temperature extremes.Over most of their growth temperature ranges the membrane lipids of wild type and temperature-sensitive B. stearothermophilus cells exist entirely or nearly entirely in the liquid-crystalline state. Also, the temperature-sensitive mutant is capable of growth at temperatures well above those at which the membrane lipid gel to liquid-crystalline phase transition is completed. Therefore, although other evidence suggests the existence of an upper limit on the degree of membrane fluidity compatible with cell growth, the phase transition upper boundary itself does not directly determine the maximum growth temperature of this organism. Similarly, the lower boundary does not determine the minimum growth temperature, since cell growth ceases at a temperature at which most of the membrane lipid still exists in a fluid state. These observations do not support the suggestion made in an earlier study, which utilized electron spin resonance spectroscopy to monitor membrane lipid lateral phase separations, that the minimum and maximum growth temperatures of this organism might be directly determined by the solid-fluid membrane lipid phase transition boundaries. Evidence is presented here that the electron spin resonance techniques used previously did not in fact detect the gel to liquid-crystalline phase transition of the bulk membrane lipids, which, however, can be reliably measured by differential thermal analysis.     

Lateral lipid distribution and phase transition in phosphatidylethanolamine/phosphatidylserine vesicles: A cross-linking study

To determine the nonideal mixing of two lipid components within the membrane, lipid cross-linking experiments were carried out on dipalmitoylphosphatidylethanolamine (DPPE) vesicles and on dipalmitoylphosphatidylethanolamine/dipalmitoylphosphatidylserine (DPPE/DPPS) vesicles. By comparison of the cross-linking reactions on both types of vesicle the mean neighbourhood relations within the binary lipid mixture can be obtained. To elucidate the relationship between cluster formation and phase transition, the temperature dependences of the lipid arrangement within the vesicle membrane and of the lipid order parameter describing the fluidity of the membrane were measured. Cluster size and phase transition correlate: during the phase transition of the lipid species with the lower phase-transition temperature (DPPS) the nonideality of the mixture increases by phase separation. Above the phase transition temperature of the second lipid species (DPPE) the clusters disappear and a slight alternating lipid arrangement is characteristic of the fluid phase.     

Interaction of trans-parinaric acid with phosphatidylcholine bilayers: comparison with the effect of other fluorophores

The effect of the fluorophore trans-parinaric acid on the structure of lipid bilayer was studied and compared with the effect of other 'perturbants'. These include commonly used fluorophores (diphenylhexatriene, heptadecylhydroxycoumarin, cis-parinaric acid and two fatty acids, palmitic and oleic acids). Differential scanning calorimetry (DSC) and proton nuclear magnetic resonance techniques were used to evaluate structural changes in the lipid bilayers. The thermodynamic parameters of dipalmitoylphosphatidylcholine multilamellar vesicles obtained from the DSC thermograms suggest that trans-parinaric acid differs from the other 'perturbants'. trans-Parinaric acid has the most pronounced impact on the Tm, the width (delta T1/2) and the index of asymmetry of the main gel to liquid crystalline phase transition without any effect on its transition, delta H. The presence of trans-parinaric acid in the lipid bilayer of dimyristoylphosphatidylcholine small unilamellar vesicles influences the chemical shift difference between the choline protons of phosphatidylcholine molecules present in the two leaflets of the vesicle bilayer (delta delta H). This suggests that trans-parinaric acid affects the head group packing in the bilayer. Its main effect is abolishing the major alterations in head group packing that occur through the phase transition. The above data indicate that trans-parinaric acid is concentrated in the gel phase domains, whereby it stabilizes the phase separation between the gel and liquid crystalline phases, probably by affecting lipid molecules present in the boundary regions between these two domain types.     

Direct measurement of the energetics of association between myelin basic protein and phosphatidylserine vesicles

A newly designed high-sensitivity isothermal reaction calorimetry system has been used to investigate the thermodynamics of the association between myelin basic protein and phosphatidylserine vesicles. This instrument has allowed us to measure directly the energetics of the protein-lipid interaction under various conditions. Above the phospholipid phase transition temperature the enthalpy of association is highly exothermic amounting to -160 kcal/mol of protein. Below the phospholipid phase transition temperature the enthalpy of association is exothermic at protein/lipid ratios smaller than 1/50 and endothermic at higher protein/lipid ratios. These studies indicate that the association of myelin basic protein to phosphatidylserine vesicles consists of at least two stages involving different types of binding. The first stage, at low protein/lipid ratios, involves a strong exothermic association of the protein to the membrane and the second, at high protein/lipid ratios, a weaker association probably involving attachment of the protein to the membrane surface only. In the gel phase the second binding stage is endothermic and appears to be correlated with the formation of large vesicle aggregates. This vesicle aggregation is a reversible process dependent upon the physical state of the membrane. The isothermal titration studies have been complemented with high-sensitivity differential scanning calorimetry experiments. It is shown that the dependence of the phospholipid transition enthalpy on the protein/lipid molar ratio can be expressed in terms of the different protein-membrane association enthalpies in the gel and fluid phases of the membrane.