Lysophosphatidylcholine stabilizes small unilamellar phosphatidylcholine vesicles. Phosphorus-31 NMR evidence for the "wedge" effect

Sonication of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-sn-glycero-3-phosphocholine (lysoPC, up to approximately 30 mol %) produces small unilamellar vesicles (SUV, 250-265 A diameter). Phosphorus-31 NMR of the POPC/lysoPC vesicles gives rise to four distinct peaks for POPC and lysoPC in the outer and in the inner bilayer leaflet which can be used to localize and quantify the phospholipids in both vesicle shells. Addition of paramagnetic ions (3 mM Pr3+) enhances outside/inside chemical shift differences and allows monitoring of membrane integrity by the absence of Pr3+ in the vesicle interior. 31P NMR shows that lysoPC in these highly curved POPC/lysoPC vesicles prefers the outer bilayer leaflet. LysoPC incorporation into POPC SUV furthermore causes a substantial and concentration-dependent decrease in spin-spin relaxations (T*2) of the outside POPC phosphorus signals from 55 ms for pure POPC vesicles (v1/2, 5.8 Hz) to 29.5 ms (v1/2, 10.8 Hz) for POPC/lysoPC vesicles containing 25 mol % lysoPC. Our findings are consistent with the idea of a cone-shaped lysoPC molecule which, for geometric reasons, is preferentially accommodated in the outer bilayer leaflet. LysoPC incorporation into POPC SUV restricts POPC headgroup motion and tightens phospholipid packing, but only in the outer bilayer shell.     

Differential scanning calorimetry and 31P NMR studies on sonicated and unsonicated phosphatidylcholine liposomes.

1. Phase transitions in sonicated (vesicles) and unsonicated liposomes composed of various synthetic phosphatidylcholines are monitored using differential scanning calorimetry and 31P NMR. 2. The temperature (Tc), heat content and width of the phase transition are comparable in both vesicles and liposomes prepared from 1,2-dipalmitoyl phosphatidylcholine and 1,2-dimyristoyl phosphatidylcholine. In vesicles composed of a (1 : 1) mixture of 1,2-dipalmitoyl phosphatidylcholine and 1,2-dioleoyl phosphatidylcholine phase separation occurs as in the bilayers of the unsonicated liposomes. 3. The linewidth of the 31P resonances in vesicles is not greatly dependent upon the fatty acid composition when the lipids are in the disordered liquid crystalline state (above Tc). When the lipids are in the gel state (below Tc), however, there is a marked increase in linewidth, demonstrating a reduction in motion of the phosphate group. 4. The ratio of the amounts of phosphatidylcholine present in the outside and inside monolayter of the vesicle membrane was determined with 31P NMR using Nd3+ as a non-permeating shift reagent. 5. The outside/inside ratio is dependent upon the hydrocarbon chain length. Increasing chain length gives a lower outside/inside ratio and a larger vesicle. Introduction of cis or trans double bonds in the chain influences the outside/inside ratio slightly. 6. The incorporation of cholesterol decreases the outside/inside ratio and increases the size of 1,2-dimyristoyl phosphatidylcholine vesicles. The cholesterol concentration in the outside and inside monolayer is approximately the same. The size of the 1,2-dioleoyl phosphatidylcholine vesicles is also increased by cholesterol incorporation but the outside/inside distribution is also increased, especially between 30 and 50 mol% cholesterol. In these vesicles cholesterol is asymmetrically distributed and strongly prefers the inside monolayer of the vesicle.     

Multicomponent spectra from 31P-NMR studies of the phase equilibria in the system dioleogylphosphatidylcholine-dioleoylphosphatidylthanolamine-water

The phase equilibria in mixtures of dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylethanolamine (DOPE) and water were studied by ³¹P-NMR and ²H-NMR. The chemical shift anisotropy is greater for DOPC than for DOPE (6–9 ppm in the lamellar phase). This difference can most probably be ascribed to different order parameters for the two lipid head groups. ³¹P-NMR spectra recorded from a lamellar phase formed by DOPC-DOPE-water below maximum hydration exhibit two resolved, superimposed powder spectra. The chemical shift anisotropy for both phospholipids has greater values at excess water contents than below maximum hydration, and the spectral resolution between DOPC and DOPE in the lamellar phase is strikingly diminished at excess water contents. From ³¹P-NMR spectra it is possible to observe relative differences in composition between different lipid phase existing in equilibrium. The proportion of DOPE is decreased in the lamellar phase, and is increased in the reversed hexagonal phase, when these phases exist in equilibrium.     

Phospholipid composition and organization of cytochrome c oxidase preparations as determined by 31P-nuclear magnetic resonance

The molecular organization as well as the composition of the phospholipids in cytochrome c oxidase preparations (bovine heart) were investigated by 31P-nuclear magnetic resonance. In the so-called 'lipid-rich' preparation the lipids were found to form a fluid bilayer around the enzyme since the 31P-NMR spectrum was characteristic of a fast, axially symmetric motion of the phosphate groups with a chemical shift anisotropy of delta sigma = -45 ppm. In contrast, the 'lipid-depleted' cytochrome c oxidase gave rise to a broader spectrum where the motion of the phospholipids was no longer axially symmetric. Nevertheless, the total width of the spectrum was still considerably narrower than observed for immobilized phospholipids in solid crystals. Both enzyme preparations were dissolved in 1% detergent solution and used for high-resolution 31P-NMR spectroscopy. Narrow lines of about 20 Hz linewidth were obtained for both types of enzyme preparations, and well-resolved resonances could be assigned to cardiolipin, phosphatidylethanolamin and phosphatidylcholine. The major differences between lipid-rich and lipid-depleted cytochrome c oxidase were the absolute amount of phospholipid associated with the protein and the relative contribution of the individual lipid classes to the 31P-NMR spectrum. For lipid-rich cytochrome c oxidase about 130 molecules phospholipid were bound per enzyme (approx. 11 cardiolipins, 54 phosphatidylethanolamines and 64 phosphatidylcholines). For lipid-depleted cytochrome c oxidase only 6-18 lipids were bound per enzyme (1 or 2 cardiolipins, 3-8 phosphatidylethanolamines and 2-8 phosphatidylcholines). In contrast to earlier suggestions that cardiolipin is the only remaining lipid in lipid-depleted cytochrome c oxidase, the 31P-NMR studies demonstrate that all three lipids remain associated with the protein.     

Conformation of fatty acyl chains in alpha- and beta-phosphatidylcholine and phosphatidylethanolamine derivatives in sonicated vesicles

Mono- and dimethylated derivatives constitute important intermediates in the conversion of phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in eucaryote membranes. 1H-NMR techniques were utilized to examine the conformation of the region of the fatty acyl chains that is close to the polar group in the series of alpha-phospholipids: PE, N-methyl-PE, N,N-dimethyl-PE, and PC. The same series of polar groups, but on phospholipid containing sn-1 and/or sn-3 fatty acyl chains (beta-phospholipids) were also examined. All of the phospholipids were in the form of small sonicated vesicles which are widely utilized as membrane models. The alpha-methylene group of the sn-1 and sn-2 fatty acyl chains of the alpha-phospholipids give rise to separate signals due to the non-equivalency of these chains with respect to the glycerol phosphate backbone on all alpha-phospholipids tested. Additionally, differences in the environment of the PC molecules as well as N-methyl-PE, and N,N-dimethyl-PE, but not PE itself on the inside and outside of the vesicles are reflected in the chemical shift of the alpha-methylene protons. On the other hand, all of the beta-phospholipids (including beta-PE) were found to reflect the inside/outside packing differences in their alpha-methylene groups. The bilayer packing does not induce any nonequivalence in the chemically equivalent acyl chains. In mixed micelles with detergents, beta-phospholipids showed one alpha-CH2 signal for all phospholipids. These results are consistent with a common conformational arrangement for the fatty acyl chains in all alpha-phospholipids that have been investigated no matter what aggregated form. The conformational arrangement in the beta-phospholipids is different, but again is similar for all of the compounds tested in various aggregated forms.     

Interaction of nonionic detergents with phospholipids in hepatic microsomes at subsolubilizing concentrations as studied by 31P-NMR

The effect of low concentrations of nonionic detergents with different critical micelle concentrations such as Triton X-100, Brij 35 and octylglucoside on rabbit liver microsomes is studied by means of ³¹P-NMR, ¹H-NMR, dynamic light scattering and functional investigations. Hexane phosphonic acid diethyl ester was used as a phosphorus membrane probe molecule to monitor the interaction of detergent molecules with microsomal phospholipids by ³¹P-NMR. This method is more sensitive than ³¹P-NMR of phospholipids alone and permitted the estimation of the maximum number of detergent molecules which can be incorporated in microsomes without the formation of mixed micelles outside the membrane. These membrane saturation concentrations were determined to be 0.07 (Brij 35), 0.1 (Triton X-100) and 0.4 (octylglucoside) (molar ratio of detergent/total phospholipids). Above these detergent concentrations, mixed micelles consisting of detergent and membrane constituents are formed, coexisting with the microsomes up to the membrane solubilization concentration. The results indicate a dependence of the membrane saturation concentration on the critical micelle concentration of the detergent and a preferential removal of phosphatidylcholine over phosphatidylethanolamine from the microsomes by all detergents studied.     

Application of (1)H and (31)P NMR to topological description of a model of biological membrane fusion: topological description of a model of biological membrane fusion

The process of biological membrane fusion can be analysed by topological methods. Mathematical analysis of the fusion process of vesicles indicated two significant facts: the formation of an inner, transient structure (hexagonal phase - H(II)) and a translocation of some lipids within the membrane. This shift had a vector character and only occurred from the outer to the inner layer. Model membrane composed of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) was studied. (31)P- and (1)H-NMR methods were used to describe the process of fusion. (31)P-NMR spectra of multilamellar vesicles (MLV) were taken at various temperatures and concentrations of Ca(2+) ions (natural fusiogenic agent). A (31)P-NMR spectrum with the characteristic shape of the H(II) phase was obtained for the molar Ca(2+)/PS ratio of 2.0. During the study, (1)H-NMR and (31)P-NMR spectra for small unilamellar vesicle (SUV), which were dependent on time (concentration of Pr(3+) ions was constant), were also recorded. The presence of the paramagnetic Pr(3+) ions permits observation of separate signals from the hydrophilic part of the inner and outer lipid bilayers. The obtained results suggest that in the process of fusion translocation of phospholipid molecules takes place from the outer to the inner layer of the vesicle and size of the vesicles increase. The NMR study has showed that the intermediate state of the fusion process caused by Ca(2+) ions is the H(II) phase. The experimental results obtained are in agreement with the topological model as well.     

Phospholipid asymmetry in renal brush-border membranes

The topological distribution of phospholipids between the inside and the outside of rabbit kidney brush-border membranes has been investigated by incubating membrane vesicles with sphingomyelinase, phospholipases A2 from bee venom and hog pancreas, phospholipases C and D, and trinitrobenzene sulfonate. Orientation and integrity of vesicles upon phospholipase treatment was determined by using two monoclonal antibodies recognizing an extracytoplasmic and a cytoplasmic domain, respectively, of the neutral endopeptidase (EC 3.4.24.11). It is shown that the transbilayer distribution of phospholipids is highly asymmetrical in kidney brush-border membranes: sphingomyelin accounted for 75% of the phospholipids present in the external leaflet, whereas phosphatidylethanolamine and phosphatidylserine plus phosphatidylinositol were found to comprise the majority of the inner layer of the membrane.     

Study of the interaction between the organophosphorus insecticide GA-41 and model membranes using NMR-spectroscopy

Interaction between organophosphorous insecticide GA-41 and liposomes formed from lecithin and multilamellar dispersion from lecithin and cardiolipin mixture (molar ratio is 7:3) has been studied by 1H- and 31P-NMR spectroscopy. The results obtained showed that while interacting with model membranes the GA-41 molecules intercalate into the range of hydrocarbon chains of phospholipids. At certain concentration of GA-41 its molecules induce the formation of some defects on bilayer structure, leading to permeability for Pr3+ ions.     

Effect of water soluble non-membrane proteins on the phospholipid structure

The interaction of water-soluble nonmembraneous proteins (trypsin and the basic pancreatic trypsin inhibitor (BPTI)) with soybean phospholipids was studied using multilamellar vesicles. Multilamellar vesicles were obtained from soybean lipid extracts and mixtures of individual phospholipids based on phosphatidylcholine. These mixtures contain different phospholipids: "bilayer", "nonbilayer", and negatively charged. It was shown that the content of both proteins in the complex depends on pH and the presence of negatively charged components. On the basis of this finding, the conclusion about the electrostatic nature of lipid-protein interaction was made. The structural organization of soybean phospholipids in multilamellar vesicles was studied in the presence and absence of the proteins using broad-line 31P-NMR spectroscopy. It was found that, in mixtures of phospholipids of complex composition, different types of phases coexist, and phospholipids of different classes can compensate the effects of each other. Trypsin and BPTI affect the structure of phospholipids in a similar way, inducing considerable structural changes in multilamellar vesicles of preparations containing negatively charged components in whose structure there coexisted primordially the bilayer and isotropic phases.     

A high-resolution NMR study (1H, 13C, 31P) of the interaction of paramagnetic ions with phospholipids in aqueous dispersions

¹H-, ¹³C-and ³¹P-NMR spectra of egg-yolk phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) and cosonicated mixtures of these phospholipids were obtained from ultrasonicatcd dispersions containing Pr³⁺, Eu³⁺, Gd³⁺ and Mn²⁺ ions.The differences in chemical shift values. °_n, between the “inner” and “outer” resonance signals for the different nuclei of the polar head group of egg-yolk phosphatidyl choline provide information about the average distances of the paramagnetic ion within the polar groups of the phospholipid molecules. In the Pr(²H₂O)³⁺_n/egg-yolk phosphatidylcholine system the ions are nearest to the phosphate and -CH₂CH₂ group, respectively but relatively far from the N(CH₃)₃ group of the polar head group of the lipid.The integral analysis of the¹ H-NMR spectra obtained from dispersions containing Pr³⁺ and Mn²⁺ ions enables us to calculate the number of the polar groups in both sides of the egg-yolk phosphatidylcholine bilayer, the size of the lipid vesicle and to give some features of the arrangement of the phospholipid molecules in cosonicated egg-yolk phosphatidylcliotine/ phosphatidytserine vesicles. At p²H 8.3 in PC/PS mixtures an extreme asymmetry is observed with PS preferentially in the outer side of the membrane. This side contains approximately three times more PS than PC molecules.Some comments are made concerning the quantitative integral analysis of proton-noise decoupled ³¹ P-NMR spectra as obtained from similar phospholipid mixtures by Michaelson et al. and Berden et at.     

Intracellular pH in stored erythrocytes. Refinement and further characterisation of the 31P-NMR methylphosphonate procedure

Methylphosphonate in conjunction with 31P-NMR spectroscopy was used for the measurement of transmembrane delta pH in human erythrocytes stored at 4 degrees C for up to 5 weeks in a nutrient medium. Intra- and extracellular pH was determined using calibration curves based on the pH-dependent separation between the NMR resonances of methylphosphonate and orthophosphate (Pi). A comprehensive statistical procedure is presented for the determination of the variance of NMR-based pH estimates. The entry of methylphosphonate into erythrocytes was more rapid at low pH and uptake was fully inhibited by the band 3 reagent, disodium 4,4-diisothiocyano-2,2'-disulphonic acid stilbene. The distribution ratio of methylphosphonate concentration inside and outside the cells was used to calculate the membrane potential; the analysis depends on a consideration of the Donnan equilibrium for an anion with one or two charges. Furthermore, the analysis does not depend on the pH estimates but relies solely on concentration estimates. The chemical shift of methylphosphonate was not subject to the variations associated with specific intracellular binding encountered with many other phosphorus compounds, including Pi. On the other hand, the ionic strength dependence of the chemical shift of methylphosphonate, contrary to earlier reports, is comparable in magnitude (but opposite in sign) to that of Pi.     

Topological asymmetry of phospholipid metabolism in rat erythrocyte membranes. Evidence for flip-flop of lecithin.

1. The distribution of phospholipids between inside and outside of rat erythrocyte membranes was studied by incubating the cells with phospholipase A2 from Naja naja venom and sphingomyelinase from Staphylococcus aureus. 2. Choline-containing phospholipids were found to comprise the majority of the outer layer of the membrane. 3. The incorporation of radioactive fatty acids into phospholipids occurred predominantly at the inside of the membrane. 4. Exchange of phospholipids between red cell membranes and plasma lipoproteins occurred at the outside of the membrane. 5. Indications were found for a rather slow flip-flop of lecithin across the membrane.     

Ion-binding to phospholipids. Interaction of calcium and lanthanide ions with phosphatidylcholine (lecithin).

Surface chemical and nuclear magnetic resonance (NMR) techniques have been used to study the interaction of Ca2+ and lanthanides with lecithins. With both methods positive reactions were detected at metal concentrations greater than 0.1 mM. 1H and 31P high-resolution NMR spectra obtained with single bilayer vesicles of lecithin were invariant up to Ca2+ concentrations of 0.1 M indicating that there is only a loose association between Ca2+ and the phospholipid. The weak interaction between Ca2+ and lecithin is confirmed by both surface chemical and NMR techniques showing that the packing of egg lecithin molecules present in bilayers does not change up to Ca2+ concentrations of about 0.1 M. The packing was also independent of pH between 1--10. Contradictory results have been reported in the literature concerning the question of Ca2+ binding to lecithins. The conflicting results are shown to have arisen from differences in the experimental conditions and differences in the sensitivity of the physical methods used by various authors to study Ca2+ -lecithin interactions. An estimate of the strength of binding and molecular details of the interaction were derived using paramagnetic lanthanides as isomorphous replacements for Ca2+. From the changes in chemical shifts induced in the presence of lanthanides an apparent binding constant KA approximately 30 l/mol was calculated at lanthanide concentrations greater than 10 mM. Using surface chemical methods it was shown that this KA is up to 10 times larger than that for Ca2+ binding. The complete assignment of the 1H NMR spectrum of lecithin, including the resonances from the relatively immobilized glycerol group, was determined to derive molecular details of the cation-lecithin interaction. From spin-lattice relaxation-time measurements and line broadening in the presence of GdCl3 it is concluded that the cations are bound to the phosphate group and that this is the only binding site. The absolute proton shifts induced by paramagnetic lanthanides depended on the nature of the ion, but the shift ratios standardised to the shift of the O3POCH2 (choline) signal were invariant throughout the lanthanide series indicating that the shifts are purely pseudocontact. In contrast the 31P shifts were found to contain significant contact contributions. These findings are consistent with a weak interaction and with the phosphate group being the binding site. The absolute shifts but not the shift ratios depended on the anion present indicating that the cation binding may be accompanied by binding of anions. Contrary to negatively charged phospholipids the interaction of lanthanides with lecithins was enhanced as the ionic strength was increased by adding NaCl. This was explained in terms of steric hindrance due to the extended conformation of the lecithin polar group.     

Light-induced membrane protein phosphorylation in the bovine rod outer segment. A magic angle spinning 31P-NMR study

Magic angle spinning 31P-NMR (MAS 31P-NMR) spectra of bovine rod outer segments, unphosphorylated and phosphorylated, were obtained. In the phosphorylated samples the spectra showed new resonances not assignable to phospholipids. These signals were present only when stimulation of receptor phosphorylation occurred. These resonances were not due to exogenous, soluble phosphorus-containing compounds. Limited proteolysis to remove the carboxyl-terminal region of the photoreceptor that contains the phosphorylation sites removed these resonances. The chemical shifts were in the usual range for serine phosphate and threonine phosphate. The pKa obtained from a pH titration of the 31P chemical shift was typical of serine phosphate. Therefore, these 31P-NMR resonances were assigned to the phosphorylation sites on membrane proteins in the rod outer segment disk membranes. Static 31P-NMR measurements revealed that at least some of these sites gave rise to relatively narrow resonances, indicative of considerable motional freedom of the carboxyl-terminal segment of the photoreceptor when phosphorylated. These data indicate that it is possible to study phosphorylation sites on membrane proteins using MAS 31P-NMR, and that using in vivo 31P 'spin labelling' one can study directly and selectively regions of receptors crucial to receptor function.     

Phosphorus nuclear magnetic resonance of Acholeplasma laidlawii cell membranes and derived liposomes.

1. The 129 MHz 31P-NMR spectrum of Acholeplasma laidlawii membranes is very similar to the spectrum of the derived liposomes and is a typical "solid state" spectrum in which the major contribution to the linewidth is made by the chemical shift anisotropy. From the value of the chemical shift anisotropy an order parameter of 0.15 is estimated for the lipid phosphates in both membranes. 2. The 31P-NMR spectrum of the A. laidlawii membrane is insensitive to pronase digestion of 4-60% of the membrane proteins and subsequent cytochrome C binding. These results indicate that either no strong lipid polar headgroup-protein interactions occur in the membrane or that the lipid-protein "complexes" in the membrane have a fast rotation (Tc shorter than 10(-6)S) along an axis perpendicular to the plane of the membrane. 3. Phospholipase A2 degrades all the phosphatidylglycerol in the membrane. The resulting membrane contains a phosphoglycolipid as the sole phosphorus-containing compound. The 31P-NMR spectrum of these membranes is identical to the spectrum of the native membranes suggesting a similar motion for the phosphate groups in both lipids. 4. Ca2+ binding to liposomes prepared from either the total polar lipids or the total phosphorus-containing lipids isolated from the A. laidlawii membrane does not affect the 21P-NMR spectrum. 5. The 31P-NMR spectrum of the membranes and derived liposomes, however, is sensitive to lipid phase transitions. When the membrane lipids are in the gel state a broadening of the 31P resonance occurs demonstrating that the polar head group motion in a biological membrane is more restricted below the lipid-phase transition temperature.     

31P-NMR of free and protein-bound molybdopterin guanine dinucleotide.

Molybdopterin guanine dinucleotide was studied by 31P-NMR in the free, iodoacetamide derivatized form [di(carboxamidomethyl)molybdopterin] and in the native state in the dimethyl sulfoxide reductase from Rhodobacter sphaeroides. The spectra confirm the presence of a pyrophosphate moiety in the cofactor molecule. Comparison of the spectrum of the free pterin with that of the protein-bound cofactor reveals a substantial upfield shift of the 31P resonances in the enzyme-bound form with respect to the free form. This shift is attributed to differences in the bond and torsional angles of the phosphates. The spectrum of the protein suggests significant coupling between the two phosphorus nuclei with coupling constants of approximately 200 Hz. Comparison of the 31P-NMR spectra of molybdopterin guanine dinucleotide and flavin adenine dinucleotide suggests that the two cofactors have similar conformations in both their free and protein-bound forms.     

Cytochrome c specifically induces non-bilayer structures in cardiolipin-containing model membranes

(1) The effect of cytochrome c addition on the phospholipid structure of liposomes composed of cardiolipin, phosphatidylserine, phosphatidylglycerol, phosphatidylcholine or phosphatidylethanolamine in a pure form or in mixtures was investigated by 31P-NMR and freeze-fracture techniques. (2) Cytochrome c specifically induces the hexagonal Hii phase and possibly an inverted micellar structure of part of the phospholipids in cardiolipin-containing model membranes. (3) These results are compared with the effect of Ca2+ on cardiolipin and are discussed in relation to the structure and function of the inner mitochondrial membrane.