Comparison of the enthalpy state of vesicles of different size by their interaction with α-lactalbumin

The characteristics of small unilamellar, large unilamellar and large multilamellar vesicles of dimyristoylphosphatidylcholine and their interaction with α-lactalbumin are compared at pH 4. (1) By differential scanning calorimetry and from steady-state fluorescence anisotropy data of the lipophilic probe 1,6-diphenyl-1,3,5-hexatriene it is shown that the transition characteristics of the phospholipids in the large unilamellar vesicles resemble more those of the multilamellar vesicles than of the small unilamellar vesicles. (2) The size and composition of the lipid-protein complex formed with α-lactalbumin around the transition temperature of the lipid are independent of the vesicle type used. Fluorescence anisotropy data indicate that in this complex the motions of the lipid molecules are strongly restricted in the presence of α-lactalbumin. (3) The previous data and a comparison of the enthalpy changes, $\text{ΔH}$, of the interaction of the three vesicle types with α-lactalbumin allow us to derive that the enthalpy state of the small unilamellar vesicles just below 24°C is about 24 kJ/mol lipid higher than the enthalpy state of both large vesicle types at the same temperature. The abrupt transition from endothermic to exothermic $\text{ΔH}$ values around 24°C for large vesicles approximates the transition enthalpy of the pure phospholipid     

Comparative study of an adenosine triphosphatase trigger-fused lipid vesicle and other vesicle forms of dimyristoylphosphatidylcholine

Several known forms of bilayer vesicles of dimyristoylphosphatidylcholine exhibit the gel to liquid-crystalline phase transition in the temperature range convenient for membrane enzyme reconstitution studies. This warrants a systematic investigation of their physical characteristics and their phase transition behaviors. We have employed electron microscopy, gel chromatography, 31P nuclear magnetic resonance, differential scanning microcalorimetry, and fluorescence spectroscopy to determine several physical parameters of the limiting size microvesicle (260 +/- 40 A), the larger vesicle form (900 +/- 100A) of Enoch and Strittmatter [Enoch, H. G., & Strittmatter, P. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 145], the multilamellar vesicle, and, in particular, an ATPase-trigger-fused macrovesicle (950 +/- 200 A). This latter vesicle form was produced by a spontaneous fusion of the complex of the plasma membrane ATPase of Schizosaccharomyces pombe and the lipid microvesicles at a low ratio of enzyme to vesicle concentrations, and at a low temperature (around 10 degrees C). The ATPase-trigger-fused vesicles are unilamellar and have an intact ionic permeation barrier at 30 degrees C and a gel to liquid-crystalline transition temperature at 24.4 degrees C with a transition heat of 5.64 kcal/mol. Thus, this vesicle form should be a valuable tool for studying possible proton-pumping activity of this ATPase. In contrast to data found in the literature, which show lack of the pretransition for unilamellar microvesicles, we have observed the pretransition around 15 degrees C for all the vesicle forms examined. Moreover, the transition widths of unilamellar vesicles are much broader than those of the multilamellar vesicles, suggesting that in the latter system interlayer interactions may contribute to the cooperativity of the transition.     

Phase behaviour of a diglyceride prodrug: spontaneous formation of unilamellar vesicles

A prodrug (Fig. 1(IV)) is synthesized consisting of the beta-blocker bupranolol which is covalently linked to 1, 3-dipalmitoyl-2-succinyl-glycerol. The resulting lipid-like prodrug is amphipathic and surface active. It disperses readily in H2O above 30 degrees C forming a smectic lamellar phase. This prodrug bears one positive charge at neutral pH and hence the swelling behaviour of dispersions in H2O is similar to that of charged phospholipids: the dispersions show continuous swelling with increasing water content and consequently in the excess H2O region of the phase diagram the thermodynamically most stable structure is the unilamellar vesicle. This includes oligomeric vesicles which may be defined as unilamellar vesicles containing smaller, also unilamellar vesicles entrapped in their internal aqueous compartment. The prodrug dispersions in H2O are polydisperse with vesicle sizes ranging from 0.1 micron to several micron. Sonication of these dispersions produce small unilamellar vesicles of an average size and size distribution similar to sonicated egg phosphatidylcholine dispersions. Unsonicated dispersions of the prodrug in H2O undergo reversibly sharp order-disorder transitions at 32 degrees C with an enthalpy change of delta H = 10 kcal/mol. In sonicated aqueous dispersions this phase transition is asymmetric and significantly broadened indicating that the cooperativity is markedly reduced. The peak temperature and enthalpy change of this broad transition are reduced compared to the transition observed with unsonicated dispersions. The temperature dependence of the electron spin resonance (ESR) hyperfine splitting and order parameter also reflects the order-disorder transition. From ESR spin labeling it is concluded that in sonicated dispersions the prodrug molecule is more mobile and its anisotropy of motion is reduced compared to unsonicated dispersions. This result indicates that the molecular packing in the highly curved bilayers of small unilamellar prodrug vesicles is significantly perturbed compared to bilayers of unsonicated dispersions.     

Fusogenic activity of delta-haemolysin from Staphylococcus aureus in phospholipid vesicles in the liquid-crystalline phase

A study has been conducted of the interaction of the lytic toxin delta-haemolysin with vesicles of phospholipid, using electron microscopy, fluorescence depolarisation and excimer fluorescence. The peptide is shown to be a fusogen towards phosphatidylcholine vesicles in fluid phases. In the presence of gel phase lipid, fusion between fluid and gel phases is not seen. Fluid phase lipid vesicles are fused together to form large multilamellar structures, and initial vesicle size does not appear to be important since small unilamellar vesicles and large unilamellar vesicles are similarly affected. Fusogenic activity of delta-haemolysin is compared to that of melittin. The former is a progressive fusogen for fluid phase lipid, while the latter causes vesicle fusion in a manner related to occurrence of a lipid phase transition.     

Lipid-DNA complex formation: reorganization and rupture of lipid vesicles in the presence of DNA as observed by cryoelectron microscopy.

Cryoelectron microscopy has been used to study the reorganization of unilamellar cationic lipid vesicles upon the addition of DNA. Unilamellar DNA-coated vesicles, as well as multilamellar DNA lipid complexes, could be observed. Also, DNA induced fusion of unilamellar vesicles was found. DNA appears to adsorb to the oppositely charged lipid bilayer in a monolayer of parallel helices and can act as a molecular "glue" enforcing close apposition of neighboring vesicle membranes. In samples with relatively high DNA content, there is evidence for DNA-induced aggregation and flattening of unilamellar vesicles. In these samples, multilamellar complexes are rare and contain only a small number of lamellae. At lower DNA contents, large multilamellar CL-DNA complexes, often with >10 bilayers, are formed. The multilamellar complexes in both types of sample frequently exhibit partially open bilayer segments on their outside surfaces. DNA seems to accumulate or coil near the edges of such unusually terminated membranes. Multilamellar lipid-DNA complexes appear to form by a mechanism that involves the rupture of an approaching vesicle and subsequent adsorption of its membrane to a "template" vesicle or a lipid-DNA complex.     

Interactions of pyrethroids with phosphatidylcholine bilayers: comparisons in liposomal systems exhibiting large or small radii of curvature

Pyrethroid interactions with dipalmitoyl phosphatidylcholine (DPPC) vesicles have been characterized in bilayers having large and small radii of curvature. The abilities of pyrethroids to alter the gel-fluid phase transition profiles were determined by steady state fluorescence anisotropy and phase-modulation lifetime techniques using the fluorescent probes cis- and trans-parinaric acid. Using the geometric isomers of parinaric acid as membrane probes, pyrethroids were found to lower the phase transition temperature (Tc) of DPPC large multilamellar vesicles with the same order of comparative effectiveness as previously reported using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). Permethrin had a greater depressive effect upon the Tc of DPPC in the small unilamellar vesicle (SUV) system than in the large multilamellar system. Conversely, allethrin was less effective in reducing the Tc of DPPC SUVs. The enhanced effect of permethrin in decreasing the Tc of DPPC SUVs was greatest in regions of more rigid lipid packing, as determined by trans-parinaric acid fluorescence parameters. The results indicate that changes in lipid packing configuration caused by differing bilayer radii of curvature may alter the interactive characteristics of pyrethroids with lipid membranes.     

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.     

Unusual enthalpy changes which accompany the titration of dimyristoylphosphatidylcholine vesicles with Triton X-100

The enthalpy changes which accompany the titration of 0.1% and 0.25% small unilamellar and multiameller vesicle samples of dimyristoylphosphatidylcholine with 2% Triton X-100 in 0.067 M phosphate buffer (pH 7.4) containing 0.15 M NaCl have been determined by titration calorimetry at 21 degrees C and 28 degrees C, the enthalpy change for both type of vesicles was zero within the limits of experimental error. At 21 degrees C, the multilamellar vesicle samples exhibited an enthalpy change of 1.35 +/- 0.48 and 2.47 +/- 0.98 kcal/mol dimyristoylphosphatidylcholine which was complete at a molar ratio of dimyristoylphosphatidylcholine to Triton of 3.21 +/- 0.84 and 5.77 +/- 1.05 for 0.1% and 0.25% dimyristoylphosphatidylcholine solutions, respectively. An exothermic transition of -2.39 +/- 0.30 and -2.05 +/- 0.69 kcal/mol phospholipid followed by an endothermic transition of 1.37 +/- 0.12 and 1.94 +/- 0.20 kcal/mol dimyristoylphosphatidylcholine was observed at 21 degrees C for 0.1% and 0.25% small unilamellar vesicle samples, respectively. In addition the nearly athermal association of the small unilemellar vesicle samples at 21 degrees C was observed, which may be an appropriate model for biological membrane fusion.     

Interaction of short-chain lecithin with long-chain phospholipids: characterization of vesicles that form spontaneously

Stable unilamellar vesicles formed spontaneously upon mixing aqueous suspensions of long-chain phospholipid (synthetic, saturated, and naturally occurring phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin) with small amounts of short-chain lecithin (fatty acid chain lengths of 6-8 carbons) have been characterized by using NMR spectroscopy, negative staining electron microscopy, differential scanning calorimetry, and Fourier transform infrared (FTIR) spectroscopy. This method of vesicle preparation can produce bilayer vesicles spanning the size range 100 to greater than 1000 A. The combination of short-chain lecithin and long-chain lecithin in its gel state at room temperature produces relatively small unilamellar vesicles, while using long-chain lecithin in its liquid-crystalline state produces large unilamellar vesicles. The length of the short-chain lecithin does not affect the size distribution of the vesicles as much as the ratio of short-chain to long-chain components. In general, additional short-chain decreases the average vesicle size. Incorporation of cholesterol can affect vesicle size, with the solubility limit of cholesterol in short-chain lecithin micelles governing any size change. If the amount of cholesterol is below the solubility limit of micellar short-chain lecithin, then the addition of cholesterol to the vesicle bilayer has no effect on the vesicle size; if more cholesterol is added, particle growth is observed. Vesicles formed with a saturated long-chain lecithin and short-chain species exhibit similar phase transition behavior and enthalpy values to small unilamellar vesicles of the pure long-chain lecithin prepared by sonication. As the size of the short-chain/long-chain vesicles decreases, the phase transition temperature decreases to temperatures observed for sonicated unilamellar vesicles. FTIR spectroscopy confirms that the incorporation of the short-chain lipid in the vesicle bilayer does not drastically alter the gauche bond conformation of the long-chain lipids (i.e., their transness in the gel state and the presence of multiple gauche bonds in the liquid-crystalline state).     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR, diffusion and entrapment analyses.

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1--0.4 micron with a mean diameter of 0.21 micron.     

Comparison of large unilamellar vesicles prepared by a petroleum ether vaporization method with multilamellar vesicles: ESR,diffusion and entrapment analyses

Large unilamellar vesicles, prepared by a petroleum ether vaporization method, were compared to multilamellar vesicles with respect to a number of physical and functional properties. Rotational correlation time approximations, derived from ESR spectra of both hydrophilic (3-doxyl cholestane) and hydrophobic (3-doxyl androstanol) steroid spin probes, indicated similar molecular packing of lipids in bilayers of multilamellar and large unilamellar liposomes. Light scattering measurements demonstrated a reduction in apparent absorbance of large unilamellar vesicles, suggesting loss of multilamellar structure which was confirmed by electron microscopy. Furthermore, large unilamellar vesicles exhibited enhanced passive diffusion rates of small solutes, releasing a greater percentage of their contents within 90 min than multilamellar vesicles, and reflecting the less restricted diffusion of a unilamellar system. The volume trapping capacity of large unilamellar vesicles far exceeded that of multilamellar liposomes, except in the presence of a trapped protein, soy bean trypsin inhibitor, which reduced the volume of the aqueous compartments of large unilamellar vesicles. Finally, measurement of vesicle diameters from electron micrographs of large unilamellar vesicles showed a vesicle size distribution predominantly in the range of 0.1–0.4 μm with a mean diameter of 0.21 μm.     

Cryoelectron microscopy of a nucleating model bile in vitreous ice: formation of primordial vesicles

Because gallstones form so frequently in human bile, pathophysiologically relevant supersaturated model biles are commonly employed to study cholesterol crystal formation. We used cryo-transmission electron microscopy, complemented by polarizing light microscopy, to investigate early stages of cholesterol nucleation in model bile. In the system studied, the proposed microscopic sequence involves the evolution of small unilamellar to multilamellar vesicles to lamellar liquid crystals and finally to cholesterol crystals. Small aliquots of a concentrated (total lipid concentration = 29.2 g/dl) model bile containing 8.5% cholesterol, 22.9% egg yolk lecithin, and 68.6% taurocholate (all mole %) were vitrified at 2 min to 20 days after fourfold dilution to induce supersaturation. Mixed micelles together with a category of vesicles denoted primordial, small unilamellar vesicles of two distinct morphologies (sphere/ellipsoid and cylinder/arachoid), large unilamellar vesicles, multilamellar vesicles, and cholesterol monohydrate crystals were imaged. No evidence of aggregation/fusion of small unilamellar vesicles to form multilamellar vesicles was detected. Low numbers of multilamellar vesicles were present, some of which were sufficiently large to be identified as liquid crystals by polarizing light microscopy. Dimensions, surface areas, and volumes of spherical/ellipsoidal and cylindrical/arachoidal vesicles were quantified. Early stages in the separation of vesicles from micelles, referred to as primordial vesicles, were imaged 23-31 min after dilution. Observed structures such as enlarged micelles in primordial vesicle interiors, segments of bilayer, and faceted edges at primordial vesicle peripheries are probably early stages of small unilamellar vesicle assembly. A decrease in the mean surface area of spherical/ellipsoidal vesicles was correlated with the increased production of cholesterol crystals at 10-20 days after supersaturation by dilution, supporting the role of small unilamellar vesicles as key players in cholesterol nucleation and as cholesterol donors to crystals. This is the first visualization of an intermediate structure that has been temporally linked to the development of small unilamellar vesicles in the separation of vesicles from micelles in a model bile and suggests a time-resolved system for further investigation.     

Temperature-induced fusion of small unilamellar vesicles formed from saturated long-chain lecithins and diheptanoylphosphatidylcholine

Small unilamellar vesicles which form when gel-state long-chain phosphatidylcholines are mixed with micellar short-chain lecithins undergo an increase in size as the long-chain species melts to its liquid-crystalline form. Analysis of the vesicle population with quasi-elastic light scattering shows that the particle size increases from 90-A radius to greater than 5000-A radius. Resonance energy transfer experiments show total mixing of lipid probes with unlabeled vesicles only when the Tm of the long-chain phosphatidylcholine is exceeded. This implies that the large size change represents a fusion process. Aqueous compartments are also mixed during this transition. 31P NMR analysis of the vesicle mixtures above the phase transition shows a great degree of heterogeneity with large unilamellar particles coexisting with oligo- and multilamellar structures. Upon cooling the vesicles below the Tm, the original size distribution (e.g., small unilamellar vesicles) is obtained, as monitored by both quasi-elastic light scattering and 31P NMR spectroscopy. This temperature-induced fusion of unilamellar vesicles is concentration dependent and can be abolished at lower total phospholipid concentrations. It occurs over a wide range of long-chain to short-chain ratios and occurs with 1-palmitoyl-2-stearoylphosphatidylcholine and dimyristoylphosphatidylcholine as well. Characterization of this fusion event is used to understand the anomalous kinetics of water-soluble phospholipases toward these unusual vesicles.     

The assembly factor P17 from bacteriophage PRD1 interacts with positively charged lipid membranes.

The interactions of the assembly factor P17 of bacteriophage PRD1 with liposomes were investigated by static light scattering, fluorescence spectroscopy, and differential scanning calorimetry. Our data show that P17 binds to positively charged large unilamellar vesicles composed of the zwitterionic 1-palmitoyl-2-oleoyl-phosphatidylcholine and sphingosine, whereas only a weak interaction is evident for 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles. P17 does not bind to negatively charged membranes composed of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and 1-palmitoyl-2-oleoyl-phosphatidylcholine. Our differential scanning calorimetry results reveal that P17 slightly perturbs the phase behaviour of neutral phosphatidylcholine and negatively charged multilamellar vesicles. In contrast, the phase transition temperature of positively charged dimyristoylphosphatidylcholine/sphingosine multilamellar vesicles (molar ratio 9 : 1, respectively) is increased by approximately 2.4 degrees C and the half width of the enthalpy peak broadened from 1.9 to 5.6 degrees C in the presence of P17 (protein : lipid molar ratio 1 : 47). Moreover, the enthalpy peak is asymmetrical, suggesting that lipid phase separation is induced by P17. Based on the far-UV CD spectra, the alpha-helicity of P17 increases upon binding to positively charged micelles composed of Triton X-100 and sphingosine. We propose that P17 can interact with positively charged lipid membranes and that this binding induces a structural change on P17 to a more tightly packed and ordered structure.     

Study of the interaction of an alpha-helical transmembrane peptide with phosphatidylcholine bilayer membranes by means of densimetry and ultrasound velocimetry

We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic alpha-helical transmembrane peptide, acetyl-K(2)-L(24)-K(2)-amide (L(24)), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L(24) decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L(24) in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L(24):PC=1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L(24). As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and (2)H NMR spectroscopy study of the interaction of the L(24) and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between (2)H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L(24) suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules.     

Morphology and phase behavior of two types of unilamellar vesicles prepared from synthetic phosphatidylcholines studied by freeze-fracture electron microscopy and calorimetry

Differential scanning calorimetry and freeze-fracture electron microscopy have been used to characterize the phase behavior and morphology of two types of unilamellar vesicles composed of synthetic phosphatidylcholines. The first type displayed an average diameter of roughly 100 nm and was formed by slow dilution and dialysis of octylglucoside-solubilized lipid. These large, unilamellar vesicles were termed dialyzed, octylglucoside vesicles and could be obtained as a fairly well defined and uniform population of vesicles. The second vesicle type was prepared by a unique procedure involving dialysis of deoxycholate-solubilized lipid at its pre-transition temperature. This procedure produced a much more heterogeneous distribution of vesicle sizes (500 to 4000 nm in diameter) and left some dilamellar and oligolamellar species which could not be conveniently separated from the giant, unilamellar vesicles constituting the major portion of the sample. Both populations of vesicles displayed phase behavior similar, but not identical to that of large, multilamellar vesicles (LMV). Fracture-face morphology of the gel phase was also observed to differ between the two unilamellar and the multilamellar species. LMV have previously been shown to have clear undulated or banded fracture-faces in the P beta phase, while octylglucoside vesicles are shown here to have facetted fracture-faces. Giant, unilamellar vesicles displayed a faint banded morphology similar to but less distinct than that of the LMV P beta phase. These results have demonstrated that bilayer apposition is not required to support the banded fracture-face morphology characteristic of the P beta phase but that a limiting curvature is necessary.     

The interaction of apolipoprotein A-I with small unilamellar vesicles of L-alpha-dipalmitoylphosphatidylcholine

The interaction between apolipoprotein A-I and small unilamellar vesicles of dipalmitoylphosphatidylcholine at the lipid phase transition resulted in complete release of vesicle contents at molar ratios of lipid to protein from 4000:1 down to 50:1. This indicated the existence of two types of stable complexes: a vesicular apo-A-I complex with a maximum of two to three apo-A-Is/vesicle, and a micellar complex (disc) with a stoichiometry of about 50 phosphatidylcholines/apo-A-I (mol/mol). We characterized the complexes by density gradient centrifugation, by gel filtration, and by immunoprecipitation using an anti-apo-A-I antibody. The morphology of the discs was similar to that of previously reported discs. Apo-A-I-induced release of vesicle contents was monitored by the relief of self-quenching of vesicle-encapsulated carboxyfluorescein. Using this assay we characterized the nature of the interaction between apo-A-I and phospholipid vesicles. The formation of complexes between vesicles and apo-A-I followed a two-step process; below or above the lipid phase transition temperature (Tc), apo-A-I bound to phosphatidylcholine vesicles but caused little leakage of contents. Kinetic analysis of the interaction between apo-A-I and dipalmitoylphosphatidylcholine vesicles below Tc indicated that about 1 in 500 collisions leads to a stable apo-A-I-vesicle complex. The second step involved passage of those complexes through Tc, which resulted in a very rapid transition into discs or vesicular complexes. Vesicular complexes contain apo-A-I which was no longer capable of interacting with pure lipid. Discs, on the other hand, interacted with vesicles at their phase transition.     

Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids

We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.     

Interaction between the 35 kDa apolipoprotein of pulmonary surfactant and saturated phosphatidylcholines. Effects of temperature

We studied the interaction between the 35 kDa apolipoprotein of canine pulmonary surfactant (SP 35) and five saturated phosphatidylcholines: distearoyl (DSPC), diheptadecanoyl (DHPC), dipalmitoyl (DPPC), dimyristoyl (DMPC), and dilauroyl (DLPC); and two monoenoic unsaturated phosphatidylcholines: dioleoyl (DOPC) and dielaidyl (DEPC), using temperatures at which all of the phospholipids except DOPC were in both the gel and liquid-crystalline states. The experiments were carried out in a buffer without Ca2+. The amount of apolipoprotein which was bound by both small unilamellar and multilayered vesicles of these lipids decreased as the temperature was increased. Moreover, near the temperatures of the phase transitions of all lipids except DLPC, there was an abrupt and marked reduction in binding of protein, in that over a 3-4 degree change in temperature there was an abrupt decrease in bound apolipoprotein. A similar change in binding occurred using DLPC, although the relatively large changes in bound protein occurred at about 10 and 20 degrees C, temperatures which are above the phase transition temperature of this lipid. Experiments using DOPC were limited to temperatures above the phase transition, and apolipoprotein binding was low. Experiments monitoring the intrinsic fluorescence of the protein, and the fluorescence of bis-1-anilino-8-naphthalene sulfonic acid bound to the protein, revealed a possible conformational change at about 40 degrees C. Measurement of intrinsic fluorescence provided the same result whether or not the protein was associated with lipid. DSC of the apolipoprotein indicated that this change was not associated with a measurable thermogenic process. We found that the interaction with DPPC was reversible at 42 degrees C, and we measured the thermodynamic parameters of the interaction at this temperature. These were: delta G0 = -8.0 kcal/mol apolipoprotein; delta H0 = -88 kcal/mol; delta S0 = -254 cal/Cdeg per mol. We conclude that the interaction between SP 35 and saturated phosphatidylcholines is temperature sensitive, and this probably reflects differences in the ability of gel and liquid-crystalline phospholipids to bind this protein. Both the delta H0 and delta S0 of the interaction are negative, and may reflect an immobilization of phospholipid around the apolipoprotein to form a boundary layer. This hypothesis is consistent with the findings obtained by DSC, in which the enthalpy of the phase transition of DMPC in lipid-apolipoprotein recombinants was found to be about 60% of that expected for a pure and unperturbed multilamellar dispersion.     

Enthalpy-driven apolipoprotein A-I and lipid bilayer interaction indicating protein penetration upon lipid binding

The interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without free cholesterol (FC) was studied by isothermal titration calorimetry and circular dichroism spectroscopy. Parameters reported are the affinity constant (K(a)), the number of protein molecules bound per vesicle (n), enthalpy change (DeltaH degrees), entropy change (DeltaS degrees ), and the heat capacity change (DeltaC(p) degrees). The binding process of apoA-I to SUVs of POPC plus 0-20% (mole) FC was exothermic between 15 and 37 degrees C studied, accompanied by a small negative entropy change, making enthalpy the main driving force of the interaction. The presence of cholesterol in the vesicles increased the binding affinity and the alpha-helix content of apoA-I but lowered the number of apoA-I bound per vesicle and the enthalpy and entropy changes per bound apoA-I. Binding affinity and stoichiometry were essentially invariant of temperature for binding to SUVs of POPC/FC at a molar ratio of 6/1 at (2.8-4) x 10(6) M(-1) and 2.4 apoA-I molecules bound per vesicle or 1.4 x 10(2) phospholipids per bound apoA-I. A plot of DeltaH degrees against temperature displayed a linear behavior, from which the DeltaC(p) degrees per mole of bound apoA-I was calculated to be -2.73 kcal/(mol x K). These results suggested that binding of apoA-I to POPC vesicles is characterized by nonclassical hydrophobic interactions, with alpha-helix formation as the main driving force for the binding to cholesterol-containing vesicles. In addition, comparison to literature data on peptides suggested a cooperativity of the helices in apoA-I in lipid interaction.