UV immobilized phospholipid bilayers

Dinitrophenyl phosphotidylethanolamine-containing bilayers have been immobilized on carbon-shadowed support films by UV irradiation of the first monolayer transferred to the support film. The immobilized bilayer is capable of allowing bound protein (anti-DNP antibody) to organize into 2-D arrays in the presence of organic solvents such as acetonitrile and dilute concentrations of detergents such as beta-octyl glucoside. The ability of the bilayers to remain attached to supports under various conditions that include organic solvents and detergents as well as divalent ions is of potential interest in the study of protein crystallization and particularly in the study of membrane proteins.     

Clustering of fatty acids in phospholipid bilayers.

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at pH greater than 11 is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Temperature-dependent perturbation of phospholipid bilayers by dimethylsulfoxide.

Dimethylsulfoxide (DMSO) is known to protect isolated enzymes during freezing while destabilizing proteins at high temperatures. This apparent paradox is the subject of a review by Arakawa et al. ((1990) Cryobiology 27, 401-415), who present evidence for a temperature-dependent, hydrophobic interaction between DMSO and non-polar moieties of proteins. The present study investigates the interaction of DMSO with phospholipid bilayers. Phospholipid vesicles containing carboxyfluorescein were exposed to several concentrations of DMSO at various temperatures. Leakage rates increased with DMSO concentration and temperature. This effect was not reduced in the presence of solutes that have been shown to neutralize DMSO toxicity in tissues. The increased leakage rates correlate well with the increased partitioning of DMSO from water to octanol at higher temperatures. Additionally, reductions in the CH2 vibrations of the bilayer are also shown to depend on DMSO concentration and temperature. A similar reduction in CH2 vibrations was observed in solutions of octanol and DMSO, suggesting that this effect is not mediated through an interaction with water. Furthermore, investigation of sulfoxide vibrations indicate that DMSO is not hydrogen bonded to the alcohol moiety of octanol, and therefore the interaction between DMSO and octanol is most likely due to a hydrophobic association. These results are consistent with a destabilization of phospholipid membranes at higher temperatures due to a hydrophobic association between DMSO and the bilayer.     

Calorimetric detection of curvature strain in phospholipid bilayers.

Phospholipids in biological membranes are arranged as bilayers. When constrained to pack into planar bilayers, certain phospholipids will form unstable structures as a consequence of their molecular shape and noncovalent bonding. This produces curvature strain which may provide energy for certain membrane processes. We demonstrate that an exothermic process associated with the relief of curvature strain can be detected calorimetrically. The enthalpy for the incorporation of a few percent lysophosphatidylcholine into large unilamellar vesicles of monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic but it is endothermic for stable bilayers such as this same lipid at pH 9 or dioleoylphosphatidylcholine at pH 7.4 or 9. The addition of lysophosphatidylcholine to monomethyldioleoylphosphatidylethanolamine at pH 7.4 is exothermic only for the addition of the first few percent of lysophosphatidylcholine and then it becomes endothermic. The size of the exothermic heat change is sensitive to changes in temperature, while the endothermic processes are relatively temperature-insensitive. The exothermic heat is also larger when 1 or 2 mol % of diolein is incorporated into vesicles of monomethyldioleoylphosphatidylethanolamine. These results are all consistent with the exothermic process corresponding to the relief of curvature strain in bilayers having a tendency to convert to the hexagonal phase. It provides a demonstration that considerable energy may be released upon the incorporation of certain molecules into membranes which have a low radius of spontaneous curvature.     

Supported planar bilayers from hexagonal phases

In this work the presence of inverted hexagonal phases H_II of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca²⁺ were observed via ³¹P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H_II phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being ∼ 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H_II phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates (∼ 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H_II phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.     

Supported planar bilayers from hexagonal phases

In this work the presence of inverted hexagonal phases H(II) of 1-palmitoy-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) and cardiolipin (CL) (0.8:0.2, mol/mol) in the presence of Ca(2+) were observed via (31)P-NMR spectroscopy. When suspensions of the same composition were extended onto mica, H(II) phases transformed into structures which features are those of supported planar bilayers (SPBs). When characterized by atomic force microscopy (AFM), the SPBs revealed the existence of two laterally segregated domains (the interdomain height being approximately 1 nm). Cytochrome c (cyt c), which binds preferentially to acidic phospholipids like CL, was used to demonstrate the nature of the domains. We used 1-anilinonaphtalen-8-sulfonate (ANS) to demonstrate that in the presence of cyt c, the fluorescence of ANS decreased significantly in lamellar phases. Conversely, the ANS binding to H(II) phases was negligible. When cyt c was injected into AFM fluid imaging cells, where SPBs of POPE:CL had previously formed poorly defined structures, protein aggregates ( approximately 100 nm diameter) were ostensibly observed only on the upper domains, which suggests not only that they are mainly formed by CL, but also provides evidence of bilayer formation from H(II) phases. Furthermore, a model for the nanostructure of the SPBs is herein proposed.     

Depolarization of dehydroergosterol in phospholipid bilayers

The behavior in phospholipid bilayers of low concentrations of dehydroergosterol, a fluorescent cholesterol mimic, has been examined by fluorometry and calorimetry. In contrast to many fluorescent membrane probes, dehydroergosterol shows a decrease in fluorescence anisotropy when the matrix phospholipid goes from the liquid-crystalline to the gel state. This was observed in three systems in which the matrix lipid was either dipalmitoyl- or dimyristoylphosphatidylcholine or dilauroylphosphatidylethanolamine. The decrease in anisotropy is the result of a large increase in the fluorescence life time of dehydroergosterol in these bilayer systems which is probably the result of thermal quenching of dehydroergosterol by neighboring molecules. The rotation of dehydroergosterol in these bilayers can be described in terms of the thermal coefficient of frictional resistance offered by the environment (Weber et al. (1984) Biochemistry 23, 6785-6788). The thermal coefficients are observed to change abruptly at the onset and completion temperatures of the gel to liquid-crystalline phase transition temperatures of the three matrix phospholipids. These changes are, however, much smaller than are the corresponding changes in the thermal coefficient observed for the fluorescent probe diphenylhexatriene in dilauroylphosphatidylethanolamine bilayers. The difference in behavior of the two fluorescent probes may be the result of lateral phase separation of dehydroergosterol similar to that reported for cholesterol in similar systems.     

Perturbation of phospholipid bilayers by DDT

The localization of the effects of DDT (5–50 mol%) addition on the acyl chain dynamics in unilamellar vesicles of two phosphatidylcholines (DPPC and egg PC) has been investigated by steady-state fluorescence polarization of a series of n-(9-anthroyloxy) fatty acids (n = 2, 6, 9, 12 and 16) whose fluorophore is located at a graded series of depths from the surface to the centre of the bilayer. The results show that DDT is a fluidizer of DPPC and egg PC bilayers. The increase in microviscosity of DPPC bilayers at 23°C begins at the centre of the bilayer (5 mol% DDT) and proceeds outward to the surface with increasing concentration of DDT (17 mol%). This pattern of effects is not evident in fluid bilayers of DPPC at 54°C or egg PC at 23°C. DDT (33 mol%) also lowers the phase transition temperature of DPPC bilayers by approximately 2 Cdeg. DDT (17 mol%) had no effect on the mean excited fluorescence life-time of 2-AP and 12-AS in DPPC, DOPC and egg PC bilayers. No quenching of 2-AP fluorescence was evident.     

Interactions between ubiquinones and phospholipid bilayers. A spin-label study.

The effect of two ubiquinones of different side chain length (Q-3; Q-9), on the fluidity of phospholipid vesicles has been investigated using stearic acid spin labels. While both oxidized quinones have a disordering effect on the lipid bilayers, the reduced forms behave in an opposite way, in that Q-3 enhances and Q-9 decreases the order of the bilayer. The ordering effect of reduced Q-3 and the attendant decreased motional freedom in the bilayer might be the result of the insertion and stacking of the quinone between the phospholipid molecules in the bilayer. Such insertion might be related to the incapability of short-chain quinones in restoring NADH oxidation in Q-depleted mitochondria.     

Intrinsic molecules in fluid phospholipid bilayers. Fluorescence probe studies

Fluorescence probe data using 1,6-diphenyl-1,3,5-hexatriene for various concentrations of intrinsic molecules (cholesterol, gramicidin A amd cytochrome oxidase) within fluid lipid bilayers have been examined. The polarization value increases with increasing concentration of intrinsic molecule and then approaches a limiting value. Empirical curve-fitting of the experimental data, change of polarization with concentration, shows that each system can be fitted approximately by an exponential curve. A theory has been constructed based upon the assumption that only one intrinsic molecule need be adjacent to a fluorescent probe molecule to affect its motion drastically. The change in probe motion then depends upon the probability p of all positions next to a lipid chain being free of intrinsic molecules. The value of the probability p has been calculated and it is shown that (formula: see text) depending on whether the intrinsic molecule spans the lipid bilayer or not. The approximation p = e-Mx gives a good fit to the data for all x, thereby explaining the observed phenomenological fit. The fluorescent probe data is interpreted to show that protein-protein contacts increase as the intrinsic protein concentration increases within the lipid bilayer. An apparent dichotomy between the results from the fluorescence probe and from the deuterium magnetic resonance is explained in terms of a dominant affect on the probe being its hindrance to motion by interaction with the intrinsic molecule (protein) whilst individual C2H2 groups of the chain may exhibit greater disorder.     

Structure and interactive properties of highly fluorinated phospholipid bilayers.

Because liposomes containing fluoroalkylated phospholipids are being developed for in vivo drug delivery, the structure and interactive properties of several fluoroalkylated glycerophosphocholines (PCs) were investigated by x-ray diffraction/osmotic stress, dipole potential, and hydrophobic ion binding measurements. The lipids included PCs with highly fluorinated tails on both alkyl chains and PCs with one hydrocarbon chain and one fluoroalkylated chain. Electron density profiles showed high electron density peaks in the center of the bilayer corresponding to the fluorine atoms. The height and width of these high density peaks varied systematically, depending on the number of fluorines and their position on the alkyl chains, and on whether the bilayer was in the gel or liquid crystalline phase. Wide-angle diffraction showed that in both gel and liquid crystalline bilayers the distance between adjacent alkyl chains was greater in fluoroalkylated PCs than in analogous hydrocarbon PCs. For interbilayer separations of less than about 8 A, pressure-distance relations for fluoroalkylated PCs were similar to those previously obtained from PC bilayers with hydrocarbon chains. However, for bilayer separations greater than 8A, the total repulsive pressure depended on whether the fluoroalkylated PC was in a gel or liquid-crystalline phase. We argue that these pressure-distance relations contain contributions from both hydration and entropic repulsive pressures. Dipole potentials ranged from -680 mV for PCs with both chains fluoroalkylated to -180 mV for PCs with one chain fluoroalkylated, compared to +415 mV for egg PC. The change in dipole potential as a function of subphase concentration of tetraphenyl-boron was much larger for egg PC than for fluorinated PC monolayers, indicating that the fluorine atoms modified the binding of this hydrophobic anion. Thus, compared to conventional liposomes, liposomes made from fluoroalkylated PCs have different binding properties, which may be relevant to their use as drug carriers.     

Capacitance--voltage relationship in phospholipid bilayers containing gangliosides

Changes in the position of the minimum of the parabolic capacitance-voltage curve allow the measurement of the amount of ganglioside present in artificial bilayers made with phosphatidylcholine-ganglioside mixtures and asymmetrically shielded with Ca2+. The screening effect of the ionic solution must be considered. With ganglioside/phospholipid molar ratios of up to 15%, all glycolipids can be found at the membrane surfaces.     

Synaptotagmin perturbs the structure of phospholipid bilayers

Synaptotagmin I (syt), an integral protein of the synaptic vesicle membrane, is believed to act as a Ca2+ sensor for neuronal exocytosis. Syt's cytoplasmic domain consists largely of two C2 domains, C2A and C2B. In response to Ca2+ binding, the C2 domains interact with membranes, becoming partially embedded in the lipid bilayer. We have imaged syt C2AB in association with lipid bilayers under fluid, using AFM. As expected, binding of C2AB to bilayers required both an anionic phospholipid [phosphatidylserine (PS)] and Ca2+. C2AB associated with bilayers in the form of aggregates of varying stoichiometries, and aggregate size increased with an increase in PS content. Repeated scanning of bilayers revealed that as C2AB dissociated it left behind residual indentations in the bilayer. The mean depth of these identations was 1.81 nm, indicating that they did not span the bilayer. Individual C2 domains (C2A and C2B) also formed aggregates and produced bilayer indentations. Binding of C2AB to bilayers and the formation of indentations were significantly compromised by mutations that interfere with binding of Ca2+ to syt or reduce the positive charge on the surface of C2B. We propose that bilayer perturbation by syt might be significant with respect to its ability to promote membrane fusion.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

The interaction of N-oleylethanolamine with phospholipid bilayers

Long chain acylamides of ethanolamine were previously found to increase in the infarcted canine myocardium. Subsequent in vitro experiments established a number of interesting biological and physiological properties of these compounds including alteration of rabbit skeletal sarcoplasmic reticulum function and inhibition of permeability dependent calcium release from heart mitochondria. These results suggested an interaction between the N-acylethanolamines and biological membranes. In the present work we show that the most potent species in previous studies, N-oleylethanolamine, forms stable complexes with phospholipid vesicles, lowers diphenylhexatriene polarization ratios in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine uni- and multilamellar bilayer vesicles, and also produces a concentration dependent decrease in the phase transitions of these lipid structures. In addition studies with parinaric acids also suggested that N-oleylethanolamine partitions preferentially into more fluid areas of the bilayer. The results are discussed in terms of possible effects on biological membranes.