Modulation of folding and assembly of the membrane protein bacteriorhodopsin by intermolecular forces within the lipid bilayer.

Three different lipid systems have been developed to investigate the effect of physicochemical forces within the lipid bilayer on the folding of the integral membrane protein bacteriorhodopsin. Each system consists of lipid vesicles containing two lipid species, one with phosphatidylcholine and the other with phosphatidylethanolamine headgroups, but the same hydrocarbon chains: either L-alpha-1, 2-dioleoyl, L-alpha-1,2-dipalmitoleoyl, or L-alpha-1,2-dimyristoyl. Increasing the mole fraction of the phosphatidylethanolamine lipid increases the desire of each monolayer leaflet in the bilayer to curve toward water. This increases the torque tension of such monolayers, when they are constrained to remain flat in the vesicle bilayer. Consequently, the lateral pressure in the hydrocarbon chain region increases, and we have used excimer fluorescence from pyrene-labeled phosphatidylcholine lipids to probe these pressure changes. We show that bacteriorhodopsin regenerates to about 95% yield in vesicles of 100% phosphatidylcholine. The regeneration yield decreases as the mole fraction of the corresponding phosphatidylethanolamine component is increased. The decrease in yield correlates with the increase in lateral pressure which the lipid chains exert on the refolding protein. We suggest that the increase in lipid chain pressure either hinders insertion of the denatured state of bacterioopsin into the bilayer or slows a folding step within the bilayer, to the extent that an intermediate involved in bacteriorhodopsin regeneration is effectively trapped.     

Non-covalent cross-linking of lipid bilayers by myelin basic protein. A possible role in myelin formation

Myelin basic protein associates with bilayer vesicles of pure egg phosphatidylcholine, l-α-dimyristoyl phosphatidylcholine and dl-α-dipalmitoyl phosphatidylcholine. Under optimum conditions the vesicles contain 15–18% of protein by weight. The binding to dipalmitoyl phosphatidylcholine is facilitated above its gel-to-liquid crystalline transition temperature. At low ionic strength the protein provokes a large increase in vesicle size and aggregation of these enlarged vesicles. Above a sodium chloride concentration of 0.07 M vesicle fusion is far less marked but aggregation persists. The pH- and ionic strength-dependence of this aggregation follows that of the protein alone; in both cases it occurs despite appreciable electrostatic repulsion between the associating species.A similar interaction was observed with diacyl phosphatidylserine vesicles.These observations, which contrast with earlier reports in the literature of a lack of binding of basic protein to phosphatidylcholine-containing lipids, demonstrate the ability of this protein to interact non-ionically with lipid bilayers. The strong cross-linking of lipid bilayers suggests a role for basic protein in myeling, raising the possibility that the protein is instrumental in collapsing the oligodendrocyte cell membrane and thus initiating myelin formation.     

Molecular packing of cholesterol in phospholipid vesicles as probed by dehydroergosterol

Ergosta-5,7,9,22-tetraen-3-β-ol (dehydroergosterol) was synthesized and employed as a probe of cholesterol behavior in phospholipid bilayers. Circular dichroism (CD) spectra were obtained. The CD of dehydroergosterol in sonicated egg phosphatidylcholine vesicles was dependent on cholesterol concentration, while in unsonicated egg phosphatidylcholine liposomes and in vesicles obtained by oxctylglucoside dialysis, the CD observed was independent of cholesterol content. The CD of dehydroergosterol in sonicated sphingomyelin vesicles exhibited a different dependence on cholesterol content than seen in sonicated egg phosphatidylcholine vesicles. These data are interpreted in terms of differences between the packing of cholesterol in systems of large and small radii of curvature and in different interactions between dehydroergosterol and phosphatidylcholine and sphingomyelin.     

Shape changes in human erythrocytes induced by replacement of the native phosphatidylcholine with species containing various fatty acids

Phosphatidylcholine-specific transfer protein from beef liver has been used to replace native phosphatidylcholine (PC) molecules from intact human erythrocytes by a variety of PC species differing in fatty acid composition. These replacements changed neither the total phospholipid content of the membrane, nor the composition of this fraction in terms of the various phospholipid classes. The morphology of the erythrocyte was not modified when native PC was replaced by 1-palmitoyl,2-oleoyl PC, 1-palmitoyl,2-linoleoyl PC, egg PC, or PC isolated from rat liver microsomes. Replacement with the disaturated species 1,2-dimyristoyl PC, 1,2-dipalmitoyl PC, and 1,2-distearoyl PC resulted in the formation of echinocytes and, at higher levels of replacement, in spheroechinocytes. Echinocyte-like erythrocytes were also observed after replacement with 1-palmitoyl,2-arachidonoyl PC, whereas stomatocytes were formed upon replacement with PC species containing two unsaturated fatty acids, e.g., 1,2-dioleoyl PC and 1,2-dilinoleoyl PC. The observations show that the erythrocyte membrane structure and the overall discoid cell shape of the human erythrocyte are optimally stabilized by PC species that contain one saturated and one mono- or diunsaturated fatty acid, and that the cell tolerates only limited variations in the species composition of its PC.     

Factors affecting the motion of the polar headgroup in phospholipid bilayers. A 31P NMR study of unsonicated phosphatidylcholine liposomes.

(1) The 129 MHZ and 36.4 MHZ 31 P NMR spectra of unsonicated liposomes consisting of phosphatidylcholines of varying chain length and unsaturation have been investigated. (2) In the liquid crystalline state the 31 P NMR liposome spectra are similar for both saturated and unsaturated phosphatidylcholines, demonstrating that the motion of the polar headgroup is not sensitive to the fatty acid composition in the disordered liquid crystalline state. (3) Below the hydrocarbon phase transition temperature there is a marked increase in the linewidth of the 31P NMR liposome spectra, indicating a reduction in the motion of the polar headgroup. (4) The addition of equimolar concentrations of cholesterol to phosphatidylcholine eliminates phase transition effects experienced by the polar headgroup. The motion of the polar headgroup is then very similar to that obtained in the liquid crystalline state for pure phosphatidylcholine bilayers. (5) In the liquid crystalline state the motion of the polar headgroup in the phosphate region is insensitive to changes in the available area per phosphatidy-choline molecule.     

NMR studies on phospholipid bilayers. Some factors affecting lipid distribution.

1. 1H-NMR and 31P-NMR are used to measure the outside/inside distribution of phospholipids in mixed vesicles. 2. Ferricyanide is a suitable shift reagent for measuring the outside/inside ratio of lecithin using 1H-NMR even when the phospholipid mixture contains negative lipids. 3. 31P-NMR can be used to measure the distribution of all phospholipids present provided the resonances are separated. 4. At 36.4 MHz the inside and outside phosphorus in lecithin vesicles have different chemical shifts. The separation at room temperature is 4-5 Hz and the individual linewidths are about 4Hz. 5. In a mixture of lecithin with phosphatidylethanolamine the latter has preference for the inside layer of the bilayer. The same holds for mixtures of lecithin with phosphatidylserine, phosphatidylinositol and phosphatidic acid. 6. In mixtures of lecithin and phosphatidylserine the preference of the latter for the inside is increased at lower pH under which conditions the negative charge of the phosphatidylserine is decreased. 7. In mixtures of lecithin with sphingomyelin the lecithin has a higher concentration at the inside. 8. The effect of vesicle size on the 31P-NMR linewidth and the temperature dependence of this linewidth is in agreement with the conclusion of Berden et al. (FEBS Lett. (1974), 46, 55-58) that the chemical shift anisotropy, modulated by the isotropic tumbling of the vesicles, makes a contribution to the linewidth. The chemical shift difference between outside and inside phosphorus can be used as a parameter for the measurement of the packing density at the inside and of the size of the vesicles. 9. It is concluded that both charge and the packing properties of the head group are major factors in determining the distribution of phospholipids in mixed vesicles.     

Liposome-mediated delivery of ribosome inactivating proteins to cells in vitro

This study describes the liposome-mediated delivery of toxins to a variety of cells in vitro. Gelonin, a potent inhibitor of protein synthesis from Gelonium multiflorum, was delivered to the cytoplasm of TLX5 lymphoma cells most effectively by phosphatidylserine vesicles. These liposomes were also capable of inhibiting protein synthesis in XC (transformed rat fibroblasts) and phytohaemagglutinin-stimulated CBA mouse lymphocytes. Phosphatidylcholine liposomes had no capacity to deliver their contents to the cytoplasm, but the addition of cholesterol to the vesicle membrane resulted in an increased capacity. Delivery events were enhanced further by the addition of mixed bovine brain gangliosides to the membrane in the ratio 5:5:1 phosphatidylcholine/cholesterol/gangliosides. The addition of cholesterol to phosphatidylserine vesicles failed to increase the inhibitory effects of the gelonin liposomes. The A chain of diphtheria toxin encapsulated in phosphatidylserine liposomes had no inhibitory effect on the level of protein synthesis in TLX5 or Daudi cells.     

Stability of small unilamellar liposomes in serum and clearance from the circulation: The effect of the phospholipid and cholesterol components

Small unilamellar liposomes containing carboxyfluorescein (CF) and composed of various unsaturated and saturated phospholipids with or without cholesterol were incubated in the presence of mouse serum at 37°C. Liposomes composed of egg L-α-phosphatidylcholine (PC), L-α-dioleoylphosphatidylcholine (DOPC) or sphingomyelin (SM) became rapidly permeable to entrapped CF but incorporation of cholesterol into such liposomes reduced CF leakage. Under similar conditions, CF leakage from cholesterol-free liposomes composed of saturated phospholipids of increasing fatty acid chain length was dependant on the liquid-crystalline phase transition temperature (Tc) of the phospholipid component. Thus, L-α-dilaureoylphos-phatidylcholine (DLPC), L-α-dimyristoyl phosphatidylcholine (DMPC) and L-α-dipalmitoylphosphatidylcholine (DPPC) with Tc's below or near the temperature of the incubation (37°C) released CF rapidly whereas L-α-diheptedecanoyl phosphatidylcholine (DHPC), L-α-distearoylphosphatidylcholine (DSPC) and hydrogenated egg PC (HPC) liposomes with Tc's above 37°C retained the dye quantitatively. After incorporation of cholesterol into liposomes composed of saturated phospholipids, CF release was reduced for DLPC and DMPC and increased for DPPC, DSPC, DHPC and HPC vesicles. Liposomes with or without cholesterol exhibiting greatest stability (in terms of CF retention) in the presence of serum were injected intravenously into mice and rates of clearance of quenched CF from the circulation measured. Observed clearance rates were linear and, when liposomes contained tritiated phospholipid, identical to those of the radiolabel suggesting retention of liposomal integrity in the intravascular space. However, half-lifes of liposomes ranging from 0.1 to 16 h did not correlate with the physical characteristics of their phospholipid component. After intraperitoneal injection, there was quantitative entry of quenched CF (stable liposomes) into the blood from which it was eliminated at rates corresponding to those observed after intravenous injection. These results suggest that solute retention by liposomes and their half-life in the circulation can be controlled by the appropriate manipulation of liposomal membrane fluidity and composition.     

Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts.

Purified phospholipid exchange protein from beef heart cytosol is used to accelerate the exchange of phospholipids between labeled sealed ghosts and phosphatidylcholine/cholesterol liposomes. The purified protein accelerates the transfer of phosphatidylcholine and, to a lesser degree, that of sphingomyelin, phosphatidylinositol, and lysophosphatidylcholine. The presence of exchange protein does not accelerate the exchange of phospholipids between intact red blood cells and liposomes, but 75% of the phosphatidylcholine of sealed ghosts is readily available for exchange. The remaining 25% is also exchangeable but at a slower rate. When the exchange is assayed between inside-out vesicles and liposomes, 37% of the phosphatidylcholine is readily available, and 63% is exchanged at a slower rate. These results are consistent with an asymmetric distribution of phosphatidylcholine in isolated erythrocyte membrane fractions. The sum of the forward and backward transposition of phosphatidylcholine between the inside and outside layers of sealed ghost membranes amounts to 11% per hour, and the half-time for equilibration is 2.3 h. Significatnly lower values are obtained for the inside-out vesicles (half-time for equilibration: 5.3 h). These results suggest that, during the formation of the vesicles, the asymmetry of phosphatidylcholine is partially preserved, but structural changes occur in the membrane that affect the rate of membrane transposition of phosphatidylcholine.     

Exchange of phospholipids between unilamellar vesicles of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine and plasma very low density lipoproteins.

Purified phosphatidylcholine exchange protein from bovine liver was used to exchange [14C]dipalmitoyl phosphatidylcholine from sonicated vesicles to human plasma very low density lipoproteins (VLDL). The exchange of [14C]-dipalmitoyl phosphatidylcholine for VLDL phospholipids was temperature dependent and linear with respect to time and amount of exchange protein. In the absence of the exchange protein, less than 10% of the [14C]dipalmitoyl phosphatidylcholine was transferred. At an initial weight ratio of [14C]-dipalmitoyl phosphatidylcholine vesicles to VLDL phospholipid (1.2 mg) of 2.2, the exchange protein (14 microgram) replaced 55% of the VLDL phospholipids with [14C]dipalmitoyl phosphatidylcholine in 15 min; VLDL protein and cholesterol content were unaltered. From these studies we conclude that the exchange protein is a useful method to alter the phospholipid composition of VLDL under conditions such that there is minimal perturbation of the lipoprotein.     

Effect of liposomal phospholipid composition on cholesterol transfer between microsomal and liposomal vesicles.

Preincubation of rat liver microsomal vesicles at 37 degrees C in the presence of [3H]cholesterol/phospholipid liposomes results in a net transfer of cholesterol from liposomes to microsomal vesicles. This transfer follows first-order kinetics. For similar concentrations of the donor vesicles, rates of transfer are about 6-8 times lower with cholesterol/sphingomyelin liposomes compared with cholesterol/phosphatidylcholine liposomes. Also, transfer of cholesterol from cholesterol/sphingomyelin liposomes to microsomal vesicles reveals a larger activation energy than for the process from cholesterol/phosphatidylcholine liposomes. There is a significant correlation between the amount of liposomal cholesterol transferred to microsomal vesicles during preincubation and the increase found with acyl-CoA:cholesterol acyltransferase activity in these microsomes over their corresponding controls. If, however, liposomes made solely of phospholipids are substituted for the cholesterol/phospholipid liposomes in the preincubation system containing microsomal vesicles, then the acyl-CoA:cholesterol acyltransferase activity is decreased compared with the corresponding control system. Both sphingomyelin and phosphatidylcholine liposomes are equally effective in decreasing the enzyme activity. These results offer direct kinetic evidence for the positive correlation between cholesterol and sphingomyelin found in vivo in biological membranes.     

Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking

Cell membranes have complex lipid compositions, including an asymmetric distribution of phospholipids between the opposing leaflets of the bilayer. Although it has been demonstrated that the lipid composition of the outer leaflet of the plasma membrane is sufficient for the formation of raft-like liquid-ordered (l(o)) phase domains, the influence that such domains may have on the lipids and proteins of the inner leaflet remains unknown. We used tethered polymer supports and a combined Langmuir-Blodgett/vesicle fusion (LB/VF) technique to build asymmetric planar bilayers that mimic plasma membrane asymmetry in many ways. We show that directly supported LB monolayers containing cholesterol-rich l(o) phases are inherently unstable when exposed to water or vesicle suspensions. However, tethering the LB monolayer to the solid support with the lipid-anchored polymer 1,2-dimyristoyl phophatidylethanolamine-N-[poly(ethylene glycol)-triethoxysilane] significantly improves stability and allows for the formation of complex planar-supported bilayers that retain >90% asymmetry for 1-2 h. We developed a single molecule tracking (SPT) system for the study of lipid diffusion in asymmetric bilayers with coexisting liquid phases. SPT allowed us to study in detail the diffusion of individual lipids inside, outside, or directly opposed to l(o) phase domains. We show here that l(o) phase domains in one monolayer of an asymmetric bilayer do not induce the formation of domains in the opposite leaflet when this leaflet is composed of palmitoyl-oleoyl phosphatidylcholine and cholesterol but do induce domains when this leaflet is composed of porcine brain phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and cholesterol. The diffusion of lipids is similar in l(o) and liquid-disordered phase domains and is not affected by transbilayer coupling, indicating that lateral and transverse lipid interactions that give rise to the domain structure are weak in the biological lipid mixtures that were employed in this work.     

Exchange of phosphatidylcholine between small unilamellar liposomes and human plasma high-density lipoprotein involves exclusively the phospholipid in the outer monolayer of the liposomal membrane

By making use of the capacity of phospholipase A₂ to degrade selectively the phospholipid in the outer half of the lipid bilayer of small unilamellar phospholipid/cholesterol vesicles without affecting the retention of a vesicle-encapsulated solute, we demonstrated that the exchange of phosphatidylcholine between such vesicles and human high density lipoprotein involves exclusively the phosphatidylcholine present in the outer monolayer of the vesicle membrane.     

Molecular dynamics of the local anesthetic tetracaine in phospholipid vesicles

Upon introduction into phosphatidylcholine vesicles, the 13C magnetic resonance peaks of the aromatic resonances of tetracaine are broadened while the T1 relaxation times show little change. Addition of tetracaine to vesicles containing 30% cholesterol produces a similar broadening in the 13C NMR spectrum of tetracaine. Nuclear magnetic resonance parameters of phosphatidylcholine in vesicles which are unchanged by the addition of equimolar tetracaine include 13C T1 relaxation time and 31P linewidth, T1 relaxation time, and nuclear Overhauser effect enhancement. These results are interpreted as indicating a hydrophobic interaction between hydrocarbon portions of the anesthetic and phospholipid bilayer. The rotational correlation time of tetracaine about its long axis in the vesicles has been calculated from the 13C NMR spin lattice relaxation times to be about 10(-10.3) s and is unchanged by incorporation into the phospholipid bilayer. The positively charged ammonium group of tetracaine interacts with the negatively charged phosphate group of the vesicle lipids. Using shift reagents and 31P NMR, tetracaine has been shown to displace cations from the bilayer surface, and does not undergo fast flip-flop across the vesicle bilayer.     

Competitive carotenoid and cholesterol incorporation into liposomes: effects on membrane phase transition, fluidity, polarity and anisotropy

Pure 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC) or mixed DPPC:1,2-dipalmitoyl phosphatidyletanolamine (DPPE):1,2-dipalmitoyl diphosphatidylserine (DPPS) (17:5:3) liposomes were incorporated with 5 mol% dietary carotenoids (beta-carotene, lutein and zeaxanthin) or with cholesterol (16 and 48 mol%) in the absence or presence of 15 mol% carotenoids, respectively. The carotenoid incorporation yields ranged from 0.42 in pure to 0.72 in mixed phospholipid liposomes. They decreased significantly, from 3 to 14%, in the corresponding cholesterol-doped liposomes, respectively. Highest incorporation yields were achieved by zeaxanthin and lutein in phospholipid liposomes while in cholesterol-containing liposomes, lutein was highest incorporated. The effects on membrane structure and dynamics were determined by differential scanning calorimetry, steady-state fluorescence and anisotropy measurements. Polar carotenoids and cholesterol cause similar, dose-dependent effects: ordering and rigidification revealed by broadening of the transition peak, and increase of anisotropy. Membrane hydrophobicity is determined by cholesterol content and carotenoid polarity. In cholesterol-doped liposomes, beta-carotene is less incorporated than in cholesterol-free liposomes. Our observations suggest effects of carotenoids, even at much lower effective concentrations than cholesterol (8 to 80-fold), on membrane structure and dynamics. Although they are minor constituents of animal membranes, carotenoids may act as modulators of membrane phase transition, fluidity, polarity and permeability, and therefore, can influence the membrane physiology and pathology.     

Transbilayer distribution and movement of lysophosphatidylcholine in liposomal membranes

Single bilayer vesicles were prepared by sonication of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine. Incubation with lysophospholipase results in a fast hydrolysis of 80–90% of lysophosphatidylcholine. The remaining lysophosphatidylcholine is only very slowly hydrolysed. There results are interpreted as lysophosphatidylcholine being asymmetrically distributed over the two halves of the bilayer. The slow phase of lysophosphatidylcholine hydrolysis sets an upper limit to the rate of transbilayer movement of lysophosphatidylcholine. The half time of this process at 37° C is estimated to be about 100 h. Incorporation of cholesterol in the vesicles reduces the distributional asymmetry of lysophosphatidylcholine to the extent of an outside-inside ratio of 60 : 40. [¹⁴C]Lysophosphatidylcholine introduced into the outer monolayer of such vesicles by intervesicular transfer of lysophosphatidylcholine remains virtually completely available for hydrolysis by lysophospholipases, corroborating the interpretation that transbilayer movement of lysophosphatidylcholine in these vesicles is an extremely slow process.In handshaken liposomes consisting of 5 mol% 1-palmitoyl lysophosphatidylcholine and 95 mol% egg phosphatidylcholine 15–20% of lysophosphatidylcholine is readily available for exogenous lysophospholipase. This pool may represent lysophosphatidylcholine in the outer monolayer of the liposomes.     

The action of phosphatidate phosphatase on the fatty-acid composition of safflower triacylglycerol and spinach glycerolipids

The microsomal phosphatidate phosphatase (EC 3.1.3.4) in maturing seeds of safflower (Carthamus tinctorius L.) was specific and selective for unsaturated phosphatidates. The relative order of specificity for phosphatidate molecular species was 1,2-dilinoleoyl = 1,2-dioleoyl > 1-palmitoyl-2-oleoyl > 1,2-dilauroyl = 1,2-dimyristoyl > 1,2-dipalmitoyl. The order of selectivity was similar to that of the specificity. The broad selectivity for unsaturated phosphatidate species (1,2-di-unsaturated-acyl and 1-saturated-acyl-2-unsaturatedacyl) led us to conclude that the phosphatidate-phosphatase reaction does not, or only very little, affect the fatty-acid composition of the diacylglycerol product and in turn the fatty-acid composition of triacylglycerol in safflower oil. As compared with the safflower microsomal enzyme, the chloroplast phosphatidate phosphatase of spinach (Spinacia oleracea L.) leaves showed a broader specificity. This agreed with the selectivity profile indicated by labelling patterns of phosphatidate and diacylglycerol synthesized from [¹⁴C]acetate in spinach chloroplasts (S.E. Gardiner et al. 1984, Biochem. J. 224, 637–643).Abbreviations BSA bovine serum albumin - FA fatty acid I thank Nippon Oil & Fats Co., Amagasaki, Japan, for providing pure oleic acid.