Dependence of M13 major coat protein oligomerization and lateral segregation on bilayer composition

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.     

Permeation of a beta-heptapeptide derivative across phospholipid bilayers

Based on a number of experiments it is concluded that the fluorescein labeled beta-heptapeptide fluoresceinyl-NH-CS-(S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH translocates across lipid vesicle bilayers formed from DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The conclusion is based on the following observations: (i) addition of the peptide to the vicinity of micrometer-sized giant vesicles leads to an accumulation of the peptide inside the vesicles; (ii) if the peptide is injected inside individual giant vesicles, it is released from the vesicles in a time dependent manner; (iii) if the peptide is encapsulated within sub-micrometer-sized large unilamellar vesicles, it is released from the vesicles as a function of time; (iv) if the peptide is submitted to immobilized liposome chromatography, the peptide is retained by the immobilized DOPC vesicles. Furthermore, the addition of the peptide to calcein-containing DOPC vesicles does not lead to significant calcein leakage and vesicle fusion is not observed. The finding that derivatives of the beta-heptapeptide (S)-beta(3)hAla-(S)-beta(3)hArg-(R)-beta(3)hLeu-(S)-beta(3)hPhe-(S)-beta(3)hAla-(S)-beta(3)hAla-(S)-beta(3)hLys-OH can translocate across phospholipid bilayers is supported by independent measurements using Tb(3+)-containing large unilamellar vesicles prepared from egg phosphatidylcholine and wheat germ phosphatidylinositol (molar ratio of 9:1) and a corresponding peptide that is labeled with dipicolinic acid instead of fluorescein. The experiments show that this dipicolinic acid labeled beta-heptapeptide derivative also permeates across phospholipid bilayers. The possible mechanism of the translocation of the particular beta-heptapeptide derivatives across the membrane of phospholipid vesicles is discussed within the frame of the current understanding of the permeation of certain oligopeptides across simple phospholipid bilayers.     

Structural and fusogenic properties of cationic liposomes in the presence of plasmid DNA.

The structural and fusogenic properties of large unilamellar vesicles (LUVs) composed of the cationic lipid N-[2,3-(dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) and 1,2-dioleoyl-3-phosphatidylethanotamine (DOPE) have been examined in the presence of pCMV5 plasmid and correlated with transfection potency. It is shown, employing lipid mixing fusion assays, that pCMV5 plasmid strongly promotes fusion between DOTMA/DOPE (1:1) LUVs and DOTMA/1,2-dioleoyl-3-phosphatidylcholine (DOTMA/DOPC) (1:1) LUVs such that at a cationic lipid-to-DNA charge ratio of 3.0, approximately 80% fusion is observed. The anions citrate and chloride can also trigger fusion, but at much higher concentrations. Freeze-fracture electron microscopy studies demonstrate the tendency of cationic vesicles to form clusters at low pCMV5 content, whereas macroscopic fused aggregates can be observed at higher plasmid levels. 31P NMR studies of the fused DNA-DOTMA/DOPE (1:1) complexes obtained at high plasmid levels (charge ratio 1.0) reveal narrow "isotropic" 31P NMR resonances, whereas the corresponding DOPC containing systems exhibit much broader "bilayer" 31P NMR spectra. In agreement with previous studies, the transfection potency of the DOPE-containing systems is dramatically higher than for the DOPC-containing complexes, indicating a correlation between transfection potential and the motional properties of endogenous lipids. Interestingly, it was found that the complexes could be separated by centrifugation into a pellet fraction, which exhibits superior transfection potencies, and a supernatant fraction. Again, the pellet fraction in the DOPE-containing system exhibits a significantly narrower 31P NMR resonance than the corresponding DOPC-containing system. It is suggested that the 31P NMR characteristics of complexes exhibiting higher transfection potencies are consistent with the presence of nonbilayer lipid structures, which may play a direct role in the fusion or membrane destabilization events vital to transfection.     

Lipid bilayers cushioned with polyelectrolyte-based films on doped silicon surfaces

Silicon semiconductors with a thin surface layer of silica were first modified with polyelectrolytes (polyethyleneimine, polystyrene sulfonate and poly(allylamine)) via a facile layer-by-layer deposition approach. Subsequently, lipid vesicles were added to the preformed polymeric cushion, resulting in the adsorption of intact vesicles or fusion and lipid bilayer formation. To study involved interactions we employed optical reflectometry, electrochemical impedance spectroscopy and fluorescent recovery after photobleaching. Three phospholipids with different charge of polar head groups, i.e. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to prepare vesicles with varying surface charge. We observed that only lipid vesicles composed from 1:1 (mole:mole) mixture of DOPC/DOPS have the ability to fuse onto an oppositely charged terminal layer of polyelectrolyte giving a lipid bilayer with a resistance of >100 kΩ. With optical reflectometry we found that the vesicle surface charge is directly related to the amount of mass adsorbed onto the surface. An interesting observation was that zwitterionic polar head groups of DOPC allow the adsorption on both positively and negatively charged surfaces. As found with fluorescent recovery after photobleaching, positively charged surface governed by the presence of poly(allylamine) as the terminal layer resulted in intact DOPC lipid vesicles adsorption whereas in the case of a negatively charged silica surface formation of lipid bilayers was observed, as expected from literature.     

Lowering line tension with high cholesterol content induces a transition from macroscopic to nanoscopic phase domains in model biomembranes

Chemically simplified lipid mixtures are used here as models of the cell plasma membrane exoplasmic leaflet. In such models, phase separation and morphology transitions controlled by line tension in the liquid-disordered (Ld)?+?liquid-ordered (Lo) coexistence regime have been described [1]. Here, we study two four-component lipid mixtures at different cholesterol fractions: brain sphingomyelin (BSM) or 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). On giant unilamellar vesicles (GUVs) display a nanoscopic-to-macroscopic transition of Ld?+?Lo phase domains as POPC is replaced by DOPC, and this transition also depends on the cholesterol fraction. Line tension decreases with increasing cholesterol mole fractions in both lipid mixtures. For the ternary BSM/DOPC/Chol mixture, the published phase diagram [19] requires a modification to show that when cholesterol mole fraction is >~0.33, coexisting phase domains become nanoscopic.     

Efficient encapsulation of DNA plasmids in small neutral liposomes induced by ethanol and calcium

Efficient encapsulation of DNA plasmids inside small, neutral liposomes composed of 1,2-dioleoyl-sn-phosphatidylcholine (DOPC), DOPC/DOPE (1,2-dioleoyl-sn-phosphatidylethanolamine) (1:1) and DOPC/DOPE/cholesterol (1:1:1) was achieved by the addition of ethanol and calcium chloride to an aqueous mixture of small unilamellar vesicles (SUVs) and plasmid. Following dialysis against low-salt buffer, the neutral lipid complexes (NLCs) had average effective diameters less than 200 nm and encapsulated up to 80% of the DNA. Optimum Ca(2+) and ethanol concentrations for each lipid mixture were determined by statistically designed experiments and mathematical modeling of trapping efficiency. NLCs are unilamellar, have neutral surface potentials, and retain entrapped DNA at pH 4.0 and in serum at 37 degrees C. The circulation and clearance properties of the complexes following intravenous administration in mice are similar to empty neutral liposomes, and the toxicity of NLCs are expected to be significantly reduced compared to other non-viral gene-delivery systems. The NLC encapsulation method, if it can be combined with effective targeting and endosome-release technologies to achieve efficient and tissue-specific transfection, may represent an important alternative to current systemic gene therapy approaches.     

15N NMR study of the ionization properties of the influenza virus fusion peptide in zwitterionic phospholipid dispersions

Influenza virus hemagglutinin (HA)-mediated membrane fusion involves insertion into target membranes of a stretch of amino acids located at the N-terminus of the HA(2) subunit of HA at low pH. The pK(a) of the alpha-amino group of (1)Gly of the fusion peptide was measured using (15)N NMR. The pK(a) of this group was found to be 8.69 in the presence of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). The high value of this pK(a) is indicative of stabilization of the protonated form of the amine group through noncovalent interactions. The shift reagent Pr(3+) had large effects on the (15)N resonance from the alpha-amino group of Gly(1) of the fusion peptide in DOPC vesicles, indicating that the terminal amino group was exposed to the bulk solvent, even at low pH. Furthermore, electron paramagnetic resonance studies on the fusion peptide region of spin-labeled derivatives of a larger HA construct are consistent with the N-terminus of this peptide being at the depth of the phosphate headgroups. We conclude that at both neutral and acidic pH, the N-terminal of the fusion peptide is close to the aqueous phase and is protonated. Thus neither a change in the state of ionization nor a significant increase in membrane insertion of this group is associated with increased fusogenicity at low pH.     

The membrane modulates internal proton transfer in cytochrome c oxidase

The functionality of membrane proteins is often modulated by the surrounding membrane. Here, we investigated the effect of membrane reconstitution of purified cytochrome c oxidase (CytcO) on the kinetics and thermodynamics of internal electron and proton-transfer reactions during O(2) reduction. Reconstitution of the detergent-solubilized enzyme in small unilamellar soybean phosphatidylcholine vesicles resulted in a lowering of the pK(a) in the pH dependence profile of the proton-uptake rate. This pK(a) change resulted in decreased proton-uptake rates in the pH range of ~6.5-9.5, which is explained in terms of lowering of the pK(a) of an internal proton donor within CytcO. At pH 7.5, the rate decreased to the same extent when vesicles were prepared from the pure zwitterionic lipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) or the anionic lipid 1,2-dioleoyl-sn-glycero-3-phospho(1-rac-glycerol) (DOPG). In addition, a small change in the internal Cu(A)-heme a electron equilibrium constant was observed. This effect was lipid-dependent and explained in terms of a lower electrostatic potential within the membrane-spanning part of the protein with the anionic DOPG lipids than with the zwitterionic DOPC lipids. In conclusion, the data show that the membrane significantly modulates internal charge-transfer reactions and thereby the function of the membrane-bound enzyme.     

Calcium/phosphate-induced immobilization of fluorescent phosphatidylserine in synthetic bilayer membranes: inhibition of lipid transfer between vesicles

Resonance energy transfer between 4-nitro-2,1,3-benzoxadiazole (NBD) acyl chain labeled phospholipid analogues and (lissamine) rhodamine B labeled phosphatidylethanolamine was used to monitor the rate of NBD-labeled lipid transfer between a variety of small unilamellar donor vesicles and dioleoylphosphatidylcholine (DOPC) acceptor vesicles. In the presence of appropriate concentrations of Ca2+ and phosphate, the transfer rate of NBD-phosphatidylserine (NBD-PS) from vesicles composed of lipid extracts from human red blood cells was reduced by approximately 10-fold, while the transfer rates of NBD-phosphatidylcholine, -ethanolamine, -glycerol, -N-succinylethanolamine, and -phosphatidic acid were essentially unaffected. A systematic evaluation of the lipid composition needed to facilitate the Ca2+/phosphate-induced inhibition of NBD-PS transfer revealed that the process was dependent upon the inclusion of both cholesterol and phosphatidylethanolamine (PE) in the donor vesicle population. Inhibition of NBD-PS transfer required the sequential addition of phosphate and Ca2+ to the vesicles, indicating that the combined interaction of Ca2+ and phosphate at the membrane surface was a prerequisite for inhibition to occur. Parallel experiments designed to determine the possible mechanism of this phenomenon showed that inhibition of NBD-PS transfer was not due to Ca2+-mediated phase separations or vesicle-vesicle fusion. However, the addition of Ca2+ and phosphate to vesicles composed of total red blood cell lipids or cholesterol/PE did result in their aggregation. On the other hand, aggregation per se did not seem to be responsible for the inhibition of transfer since NBD-PS-containing vesicles composed of DOPC or DOPC/DOPE also aggregated, although NBD-PS transfer was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent probe partitioning in GUVs of binary phospholipid mixtures: implications for interpreting phase behavior

The phase behavior of membrane lipids is known to influence the organization and function of many integral proteins. Giant unilamellar vesicles (GUVs) provide a very useful model system in which to examine the details of lipid phase separation using fluorescence imaging. The visualization of domains in GUVs of binary and ternary lipid mixtures requires fluorescent probes with partitioning preference for one of the phases present. To avoid possible pitfalls when interpreting the phase behavior of these lipid mixtures, sufficiently thorough characterization of the fluorescent probes used in these studies is needed. It is now evident that fluorescent probes display different partitioning preferences between lipid phases, depending on the specific lipid host system. Here, we demonstrate the benefit of using a panel of fluorescent probes and confocal fluorescence microscopy to examine phase separation in GUVs of binary mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Patch and fibril gel phase domains were found to co-exist with liquid disordered (l(d)) domains on the surface of GUVs composed of 40:60 mol% DOPC/DPPC, over a wide range of temperatures (14-25°C). The fluorescent lipid, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl (NBD-DPPE), proved to be the most effective probe for visualization of fibril domains. In the presence of Lissamine(TM) rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Rh-DPPE) we were unable to detect fibril domains. This fluorophore also affected the partitioning behavior of other fluorescent probes. Overall, we show that the selection of different fluorescent probes as lipid phase reporters can result in very different interpretation of the phase behavior of DOPC/DPPC mixtures.     

Poly(ethylene glycol)-induced lipid mixing but not fusion between synthetic phosphatidylcholine large unilamellar vesicles

We have examined the effect of poly(ethylene glycol) (PEG) on stable large unilamellar vesicles formed by a rapid extrusion technique and composed of pure synthetic phosphatidylcholines. The lipid systems studied were the saturated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and the monounsaturated 1,2-dioleoyl-sn-glycerol-3-phosphocholine (DOPC). PEG at all concentrations (3.8-40 wt %) induced lipid mixing between large vesicles composed of these phosphatidylcholines. Extensive leakage of internal contents also occurred at high PEG concentrations. However, in contrast to our previous report [Parente, R. A., & Lentz, B. R. (1986) Biochemistry 25, 6678], we could detect no mixing of internal contents indicative of fusion. This discrepancy is due to environmental factors that affect the behavior of 8-amino-naphthalene-1,3,6-trisulfonic acid (ANTS), the fluorophore used in the assay for contents mixing and leakage [McIntyre, Parks, Massenburg, & Lentz (1991) (submitted)]. In agreement with the results of the fusion assay, quasielastic light-scattering measurements revealed no increase in vesicle size following treatment with PEG. These results emphasize the importance of using assays for both membrane mixing and contents mixing to demonstrate fusion, since significant lipid mixing occurred in the absence of fusion. We conclude that large vesicles composed of pure phosphatidylcholine do not fuse in the presence of even high concentrations of PEG. However, DOPC vesicles containing a small amount of an amphipathic "impurity" have been shown to fuse in the presence of PEG at 23 degrees C. These results are discussed in terms of their implications for the mechanism of PEG-induced membrane fusion.     

Effects of hemagglutinin fusion peptide on poly(ethylene glycol)-mediated fusion of phosphatidylcholine vesicles.

The effects of hemagglutinin (HA) fusion peptide (X-31) on poly(ethylene glycol)- (PEG-) mediated vesicle fusion in three different vesicle systems have been compared: dioleoylphosphatidylcholine (DOPC) small unilamellar vesicles (SUV) and large unilamellar vesicles (LUV) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar perturbed vesicles (pert. LUV). POPC LUVs were asymmetrically perturbed by hydrolyzing 2.5% of the outer leaflet lipid with phospholipase A(2) and removing hydrolysis products with BSA. The mixing of vesicle contents showed that these perturbed vesicles fused in the presence of PEG as did DOPC SUV, but unperturbed LUV did not. Fusion peptide had different effects on the fusion of these different types of vesicles: fusion was not induced in the absence of PEG or in unperturbed DOPC LUV even in the presence of PEG. Fusion was enhanced in DOPC SUV at low peptide surface occupancy but hindered at high surface occupancy. Finally, fusion was hindered in proportion to peptide concentration in perturbed POPC LUV. Contents leakage assays demonstrated that the peptide enhanced leakage in all vesicles. The peptide enhanced lipid transfer between both fusogenic and nonfusogenic vesicles. Peptide binding was detected in terms of enhanced tryptophan fluorescence or through transfer of tryptophan excited-state energy to membrane-bound diphenylhexatriene (DPH). The peptide had a higher affinity for vesicles with packing defects (SUV and perturbed LUV). Quasi-elastic light scattering (QELS) indicated that the peptide caused vesicles to aggregate. We conclude that binding of the fusion peptide to vesicle membranes has a significant effect on membrane properties but does not induce fusion. Indeed, the fusion peptide inhibited fusion of perturbed LUV. It can, however, enhance fusion between highly curved membranes that normally fuse when brought into close contact by PEG.     

Effect of ions on the organization of phosphatidylcholine/phosphatidic acid bilayers

Lipid bilayers are two-dimensional fluids. Here, the effect of monovalent ion concentration on the mixing, and consequently the organization, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/1,2-dioleoyl-sn-glycero-3-phosphate (DOPA) bilayers has been examined. Epifluorescence microscopy was used to visualize the organization. Fluorescence recovery after photobleaching and attenuated total reflection-Fourier transform infrared spectroscopy were used to assess the fluidity of the lipids. At high ionic strength the DOPC and DOPA lipids appear uniformly mixed. Upon lowering the ionic strength, rapid separation is observed. The DOPA-rich regions appear fractal-like and exhibit hysteresis in their properties. The lipids freely exchange between the two regions. These experiments clearly demonstrate the significant effect that electrostatics can have on membrane organization.     

Interaction of dolichol and dolichyl phosphate with phospholipid bilayers

The thermotropic phase transition of dipalmitoylphosphatidylcholine vesicles reconstituted with dolichol or dolichyl phosphate was investigated as a function of the lipid-to-polyisoprenoid ratio by means of differential scanning calorimetry and fluorescence depolarization of the embedded probe 1,6-diphenyl-1,3,5-hexatriene. At the concentrations studied, dolichol and dolichyl phosphate lowered and broadened the transition temperature of dipalmitoylphosphatidylcholine bilayers. Dolichol was found to increase the motional freedom of the bilayer both below and above the transition temperature as determined by fluorescence depolarization. In contrast, low concentrations of dolichyl phosphate decreased the bilayer motional freedom below the transition temperature while high concentrations increased the motional freedom. Above the transition temperature, dolichyl phosphate decreased bilayer 'fluidity' at all concentrations. The data suggest that these polyisoprenoids perturb the bilayer lattice, with the neutral species dolichol increasing membrane 'fluidity', while dolichyl phosphate acts to 'stiffen' the membrane.     

Temperature dependence of the structural dimensions of the inverted hexagonal (HII) phase of phosphatidylethanolamine-containing membranes

The characteristic temperature dependence of the lattice basis vector length d of phospholipid-water systems in the inverted hexagonal (HII) phase has been investigated with X-ray diffraction. For 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), d falls sharply from 78.1 A at 10 degrees C to 62.5 A at 90 degrees C. When used in conjunction with the volume fractions of the constituents, d can be used to determine the dimensions within the lipid and water regions. These data showed that a reduction in the radius of the HII-phase water cylinders Rw accounted for most of the reduction in d. From geometrical relationships between the dimensions in the HII phase, it was shown that both d and Rw are sensitive functions of the thickness of the lipid monolayer dHII. The characteristic shape of d(T) could be parameterized with the small temperature dependence of dHII along with the ratio v/a, which is the ratio of the specific volume to the area per lipid molecule at the polar interface. The ratio v/a was found to be independent of temperature for the fully hydrated HII system. Additional measurements made with a mixture of DOPE and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), mole ratio 5.07:1, produced a similar parameterization of d(T). The larger basis vector lengths for this mixture compared to those for DOPE can be attributed to a smaller ratio of v/a, which was also found to be temperature independent for this mixture. The smaller value of v/a is due to the larger effective headgroup area of DOPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The dynamics of giant unilamellar vesicle oxidation probed by morphological transitions

We have studied the dynamics of Lissamine Rhodamine B dye sensitization-induced oxidation of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) giant unilamellar vesicles (GUVs), where the progression of the underlying chemical processes was followed via vesicle membrane area changes. The surface-area-to-volume ratio of our spherical GUVs increased after as little as ten seconds of irradiation. The membrane area expansion was coupled with high amplitude fluctuations not typical of GUVs in isoosmotic conditions. To accurately measure the area of deformed and fluctuating membranes, we utilized a dual-beam optical trap (DBOT) to stretch GUV membranes into a geometrically regular shape. Further oxidation led to vesicle contraction, and the GUVs became tense, with micron-scale pores forming in the bilayer. We analyzed the GUV morphological behaviors as two consecutive rate-limiting steps. We also considered the effects of altering DOPC and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (RhDPPE) concentrations. The resulting kinetic model allows us to measure how lipid molecular area changes during oxidation, as well as to determine the rate constants controlling how quickly oxidation products are formed. Controlled membrane oxidation leading to permeabilization is also a potential tool for drug delivery based on engineered photosensitizer-containing lipid vesicles.     

Nucleotide chain length and the morphology of complexes with cationic amphiphiles: (31)P-NMR observations

31P-NMR and UV spectroscopies were used to study the interactions between cationic amphiphile-containing lipid bilayers and either a phosphorothioate oligonucleotide (OligoS) (n=21) or polyadenylic acid (PolyA) (n approximately 18,000). Multilamellar vesicles (MLVs) were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in binary mixture with either of the cationic lipids, N-[1-(2, 3-dioleoyloxy)propyl]-N',N',N'-trimethylammonium chloride (DOTAP) or cetyltrimethylammonium bromide (CTAB). A UV-difference assay showed that OligoS binding ceased above a 1:1 anion/cation ratio, while PolyA binding continued until a 2:1 ratio was reached, indicating a 'flat' conformation for bound OligoS, but not necessarily for PolyA. Cross-polarization (31)P-NMR of the nucleotide chains bound to 100% DOTAP MLVs produced spectra virtually identical to those of dry powders of OligoS or PolyA, indicating effective immobilization of the surface-bound nucleotide chains. Hahn echo (31)P-NMR showed that MLVs composed of binary mixtures of POPC with DOTAP or CTAB retained a lamellar bilayer architecture upon adding nucleotide chains. At less than stoichiometric anion/cation ratios little or no signal attributable to free nucleotide chains was visible. A narrow signal at the chemical shift expected for phosphorothiodiesters or phosphodiesters became visible at greater levels of added OligoS or PolyA, respectively, indicating the presence of mobile nucleotide chains. Salt addition caused complete desorption of the nucleotide chains. When POPC was replaced with DOPE, binding of OligoS or PolyA produced non-bilayer lipid phases in the presence of DOTAP, but not in the presence of CTAB.     

Effect of glycophorin on lipid polymorphism: A 31P-NMR study

(1) The effect of glycophorin, a major intrinsic glycoprotein of the human erythrocyte membrane, on lipid polymorphism has been investigated by ³¹P-NMR (at 36.4 MHz) and by freeze-fracture electron microscopy. (2) Incorporation of glycophorin into vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) results in the formation of unilamellar vesicles (1000–5000 Å diameter) which exhibit ³¹P-NMR bilayer spectra over a wide range of temperature. A reduction in the chemical shift anisotropy (Δσ_csa^eff) and an increase in spectral linewidth in comparison to dioleoylphosphatidylcholine liposomes may suggest a decrease in phospholipid headgroup order. (3) 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), in the presence of excess water, undergoes a bilayer to hexagonal (H_II) phospholipid arrangement as the temperature is increased above 0°C. Incorporation of glycophorin into this system stabilizes the bilayer configuration, prohibiting the formation of the H_II phase. (4) Cosonication of glycophorin with DOPE in aqueous solution (pH 7.4) produces small, stable unilamellar vesicles (300–1000 Å diameter), unlike DOPE alone which is unstable and precipitates from solution. (5) The current study demonstrates the bilayer stabilizing capacity of an intrinsic membrane protein, glycophorin, most likely by means of a strong hydrophobic interaction between the membrane spanning portion of glycophorin and the hydrophobic region of the phospholipid.     

Metabolic interconversion of dolichol and dolichyl phosphate during development of the sea urchin embryo

The in vivo and in vitro synthesis and turnover of dolichol and dolichyl phosphate have been studied over the course of early development in sea urchin embryos. Synthesis of dolichol and dolichyl phosphate was studied in vivo and in vitro using [3H]acetate and [14C] isopentenylpyrophosphate, respectively, as precursors. Both the in vivo and in vitro results indicate that the principal labeled end product of de novo synthesis is the free alcohol, and that this alcohol is subsequently phosphorylated to produce dolichyl phosphate. The presence of 30 microM compactin inhibits the de novo synthesis of dolichol from [3H]acetate by greater than 90%, but has no effect on the incorporation of 32Pi into dolichyl phosphate for more than 6 h, thus suggesting that during this time interval the major source of dolichyl phosphate is preformed dolichol. The rate of turnover of the [3H]acetate-labeled polyisoprenoid backbone of dolichyl phosphate is very slow (t1/2 = 40-70 h). In contrast, the rate of loss of the [32P]phosphate headgroup is more rapid (t1/2 = 5.7-7.7 h) and increases over the course of development. Finally, dolichyl phosphate phosphatase activity has been measured in vitro. The activity of this enzyme, which can be distinguished from phosphatidic acid phosphatase, was found to increase as a function of development, in qualitative agreement with the increased turnover of 32P from dolichyl phosphate observed in vivo. These results suggest that the phosphate moiety of dolichyl phosphate is in a dynamic state, and that dolichol kinase and dolichyl phosphate phosphatase play key roles in regulating the cellular level of dolichyl phosphate.     

The structural topology of wild-type phospholamban in oriented lipid bilayers using 15N solid-state NMR spectroscopy

For the first time, 15N solid-state NMR experiments were conducted on wild-type phospholamban (WT-PLB) embedded inside mechanically oriented phospholipid bilayers to investigate the topology of its cytoplasmic and transmembrane domains. 15N solid-state NMR spectra of site-specific 15N-labeled WT-PLB indicate that the transmembrane domain has a tilt angle of 13 degrees+/-6 degrees with respect to the POPC (1-palmitoyl-2-oleoyl-sn-glycero-phosphocholine) bilayer normal and that the cytoplasmic domain of WT-PLB lies on the surface of the phospholipid bilayers. Comparable results were obtained from site-specific 15N-labeled WT-PLB embedded inside DOPC/DOPE (1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) mechanically oriented phospholipids' bilayers. The new NMR data support a pinwheel geometry of WT-PLB, but disagree with a bellflower structure in micelles, and indicate that the orientation of the cytoplasmic domain of the WT-PLB is similar to that reported for the monomeric AFA-PLB mutant.