Probing the interaction of lipids with the non-annular binding sites of the potassium channel KcsA by magic-angle spinning NMR

The activity of the potassium channel KcsA is tightly regulated through the interactions of anionic lipids with high-affinity non-annular lipid binding sites located at the interface between the channel's subunits. Here we present solid-state phosphorous NMR studies that resolve the negatively charged lipid phosphatidylglycerol within the non-annular lipid-binding site. Perturbations in chemical shift observed upon the binding of phosphatidylglycerol are indicative of the interaction of positively charged sidechains within the non-annular binding site and the negatively charged lipid headgroup. Site directed mutagenesis studies have attributed these charge interactions to R64 and R89. Functionally the removal of the positive charges from R64 and R89 appears to act synergistically to reduce the probability of channel opening.     

Response of the headgroup of phosphatidylglycerol to membrane surface charge as studied by deuterium and phosphorus-31 nuclear magnetic resonance

The response to membrane surface charge of the glycerol headgroup of dimyristoyl-phosphatidylglycerol (DMPG) was investigated via deuterium and phosphorus-31 nuclear magnetic resonance spectroscopy. The membrane surface charge was manipulated by adding various amounts of neutral dimyristoylphosphatidylcholine (DMPC) and/or positively charged didodecyldimethylammonium bromide (DDAB) to the negatively charged DMPG, selectively deuterated at the alpha and beta segments of its glycerol headgroup. The deuterium and phosphorus-31 nuclear magnetic resonance spectra were all characteristic of random dispersions of liquid-crystalline lipids in a bilayer configuration. Differential scanning calorimetry showed that all mixtures investigated exhibited gel to liquid-crystalline phase transitions below 35 degrees C. Measurements of the deuterium quadrupole splitting and of the phosphorus-31 chemical shift anisotropy lead to the following observations. (1) Dilution of the negative surface charge density by the addition of DMPC had little effect on the quadrupole splitting from either alpha- or beta-deuterated DMPG. (2) Direct cancellation of the negative surface charge density by addition of DDAB led to a progressive decrease in the quadrupole splitting measured from alpha-deuterated DMPG, while the quadrupole splitting measured from beta-deuterated DMPG increased. For alpha-deuterated DMPG addition of 0.3 mole fraction of DDAB resulted in the appearance of two distinct quadrupole splittings. No such effect was observed for beta-deuterated DMPG.     

Examining the contributions of lipid shape and headgroup charge on bilayer behavior

To better understand bilayer property dependency on lipid electrostatics and headgroup size, we use atomistic molecular dynamics simulations to study negatively charged and neutral lipid membranes. We compare the negatively charged phosphatidic acid (PA), which at physiological pH and salt concentration has a negative spontaneous curvature, with the negatively charged phosphatidylglycerol (PG) and neutrally charged phosphatidylcholine (PC), both of which have zero spontaneous curvature. The PA lipids are simulated using two different sets of partial charges for the headgroup and the varied charge distribution between the two PA systems results in significantly different locations for the Na⁺ ions relative to the water/membrane interface. For one PA system, the Na⁺ ions are localized around the phosphate group. In the second PA system, the Na⁺ ions are located near the ester carbonyl atoms, which coincides with the preferred location site for the PG Na⁺ ions. We find that the Na⁺ ion location has a larger effect on bilayer fluidity properties than lipid headgroup size, where the A_lipid and acyl chain order parameter values are more similar between the PA and PG bilayers that have Na⁺ ions located near the ester groups than between the two PA bilayers.     

Anionic lipids stimulate Sec-independent insertion of a membrane protein lacking charged amino acid side chains

We have investigated the influence of the different lipid classes of Escherichia coli on Sec-independent membrane protein insertion, using an assay in which a mutant of the single-spanning Pf3 coat protein is biosynthetically inserted into liposomes. It was found that phosphatidylethanolamine and other non-bilayer lipids do not have a significant effect on insertion. Surprisingly, the anionic lipids phosphatidylglycerol and cardiolipin stimulate N-terminal translocation of the protein, even though it has no charged amino acid side chains. This novel effect is general for anionic lipids and depends on the amount of charge on the lipid headgroup. Since the N-terminus of the protein is at least partially positively charged due to a helix dipole moment, apparently negatively charged lipids can stimulate translocation of slightly positively charged protein segments in a direction opposite to the positive-inside rule. A mechanism is proposed to explain these results.     

The interaction of the Bax C-terminal domain with negatively charged lipids modifies the secondary structure and changes its way of insertion into membranes

Fourier transform infrared spectroscopy (FTIR) was used to study the secondary structure of peptides which imitate the amino acid sequences of the C-terminal domain of the pro-apoptotic protein Bax (Bax-C) when incorporated into different lipid vesicles with or without negatively charged phospholipids. The infrared spectroscopy results showed that while the beta-sheet components are predominant in the membrane-free Bax-C secondary structure as well as in the presence of phosphatidylcholine vesicles, the peptide changes its secondary structure in the presence of negatively charged membranes, including phospholipids such as phosphatidylglycerol or phosphatidylinositol, depending on both the lipid composition and their molar ratio. The negative charges in the model membrane surface caused a marked change from beta-sheet to alpha-helix structure. Moreover, using attenuated total reflection infrared spectroscopy (ATR-FTIR), we investigated the orientation of Bax-C alpha-helical structures with respect to the normal to the internal reflection element. The orientation of Bax-C in membranes was also affected by negatively charged lipids, the presence of phosphatidylglycerol reduced the angle it forms with the normal to the germanium plate from 45 degrees in phosphatidylcholine to 27 degrees in phosphatidylglycerol vesicles. These results highlight the importance of lipid-protein interaction for the correct folding of membrane proteins and they suggest that the C-terminal domain of Bax will only span membranes with a net negative charge in their surface.     

The structure of the antimicrobial peptide Ac-RRWWRF-NH2 bound to micelles and its interactions with phospholipid bilayers.

The hexapeptide Ac-RRWWRF-NH2 has earlier been identified as a potent antimicrobial peptide by screening synthetic combinatorial hexapeptide libraries. In this study, it was found that this peptide had a large influence on the thermotropic phase behavior of model membranes containing the negatively charged headgroup phosphatidylglycerol, a major component of bacterial membranes. In contrast, differential scanning calorimetry showed that it had little effect on model membranes containing the zwitterionic phosphatidylcholine headgroup, the main component of erythrocyte membranes. This behavior is consistent with its biological activity and with its affinity to these membranes as determined by titration calorimetry, implying that peptide-lipid interactions play an important role in this process. The structure of this peptide bound to membrane-mimetic sodium dodecyl sulfate (SDS) and dodecylphosphocholine micelles has been determined using conventional two-dimensional nuclear magnetic resonance methods. It forms a marked amphipathic structure in SDS with its hydrophobic residues on one side of the structure and with the positively charged residues on the other side. This amphipathic structure may allow this peptide to penetrate deeper into the interfacial region of negatively charged membranes, leading to local membrane destabilization. Knowledge about the importance of electrostatic interactions of Arg and the role of Trp residues as a membrane interface anchor will provide insight into the future design of potent antimicrobial peptidomimetics.     

Lipid Bilayer Modules as Determinants of K+ Channel Gating

The crystal structure of the sensorless pore module of a voltage-gated K⁺ (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.     

Effects of lipid structure on peptide-lipid interactions complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon cacitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin-DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol. There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Effects of lipid structure on peptide-lipid interactions. Complexes of salmon calcitonin with phosphatidylglycerol and with phosphatidic acid

The interactions of salmon calcitonin with a number of phospholipids are studied by electron microscopy, circular dichroism and the leakage of carboxyfluorescein. At room temperature, calcitonin reacts strongly with dimyristoylphosphatidylglycerol and egg phosphatidic acid, while only moderate or no interaction is observed with several other phospholipids. The interaction is judged by the dissolution of the phospholipid dispersion and by electron microscopic observation and is in general concomitant with an increase in the helical content of the peptide. The electrostatic charge and the transition temperature of each of the phospholipids are important factors in determining the extent of reaction with salmon calcitonin. An exception is the sulphatide from bovine brain. The resulting morphology of the complex formed between salmon calcitonin and phosphatidic acid is quite different from that formed with phosphatidylglycerol. In the case of phosphatidylglycerol and most other negatively charged phospholipids, disc-shaped complexes are observed under the electron microscope by negative staining. The calcitonin- DMPG complexes are about 7 nm thick and their diameter increases with an increasing lipid-to-peptide ratio. In contrast, phosphatidic acids form spherical complexes with salmon calcitonin causing large multilamellar structures to spontaneously break-up into smaller particles of about 10 to 20 nm in diameter independent of the lipid-to-peptide ratio. The contrasting effects of salmon calcitonin on the morphology of these two phospholipids is explicable by consideration of the size of the lipid headgroup. Phosphatidic acid can accommodate the peptide without rupture of the bilayer, while the larger headgroup of phosphatidylglycerol requires the bilayer to rupture. This model is supported by studies of calcitonin-induced leakage of carboxyfluorescein from sonicated vesicles of 75% egg phosphatidylcholine and 25% either egg phosphatidic acid, egg phosphatidylglycerol or dimyristoylphosphatidylglycerol . There was a much greater increase in carboxyfluorescein leakage from phosphatidylglycerol-containing vesicles induced by salmon calcitonin demonstrating the greater ability of the peptide to rupture bilayers containing this phospholipid.     

Spontaneous vesiculation of large multilamellar vesicles composed of saturated phosphatidylcholine and phosphatidylglycerol mixtures

The influence of temperature and ionic strength on the vesiculation properties of large multilamellar vesicles containing various proportions of dimyristoylphosphatidylglycerol has been investigated. It is shown that at low ionic strengths preformed large multilamellar vesicles composed of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol (7:3) on incubation at the gel to liquid-crystalline transition temperature (Tc approximately 23 degrees C) spontaneously vesiculate to form predominantly unilamellar systems with a mean diameter of 120 nm. Such vesiculation is not observed for incubations at temperatures appreciably above or below Tc, and is also inhibited by higher ionic strengths. Stable large multilamellar vesicles are formed, however, in systems containing the dioleoyl species of phosphatidylcholine or phosphatidylglycerol and also for dimyristoylphosphatidylcholine/dimyristoylphosphatidylserine mixtures. The vesiculation properties of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol mixtures, therefore, appear to reflect an instability in the region of the Tc driven by surface potential effects which are specific for the glycerol headgroup.     

How Lipid Headgroups Sense the Membrane Environment: An Application of 14N NMR

The orientation of lipid headgroups may serve as a powerful sensor of electrostatic interactions in membranes. As shown previously by ²H NMR measurements, the headgroup of phosphatidylcholine (PC) behaves like an electrometer and varies its orientation according to the membrane surface charge. Here, we explored the use of solid-state ¹⁴N NMR as a relatively simple and label-free method to study the orientation of the PC headgroup in model membrane systems of varying composition. We found that ¹⁴N NMR is sufficiently sensitive to detect small changes in headgroup orientation upon introduction of positively and negatively charged lipids and we developed an approach to directly convert the ¹⁴N quadrupolar splittings into an average orientation of the PC polar headgroup. Our results show that inclusion of cholesterol or mixing of lipids with different length acyl chains does not significantly affect the orientation of the PC headgroup. In contrast, measurements with cationic (KALP), neutral (Ac-KALP), and pH-sensitive (HALP) transmembrane peptides show very systematic changes in headgroup orientation, depending on the amount of charge in the peptide side chains and on their precise localization at the interface, as modulated by varying the extent of hydrophobic peptide/lipid mismatch. Finally, our measurements suggest an unexpectedly strong preferential enrichment of the anionic lipid phosphatidylglycerol around the cationic KALP peptide in ternary mixtures with PC. We believe that these results are important for understanding protein/lipid interactions and that they may help parametrization of membrane properties in computational studies.     

Products of phosphatidylglycerol turnover in two Bacillus strains with and without lipoteichoic acid in the cells

In order to understand the phosphatidylglycerol turnover mechanism, especially the differential turnover of diacylated and unacylated glycerol moieties of the lipid, products of phosphatidylglycerol metabolism were surveyed in vivo in Bacillus subtilis W23 and an alkalophile, Bacillus sp. strain A007. When cells of B. subtilis W23 labeled with radioactive glycerol were chased, lipoteichoic acid accumulated 90% of the radioactivity lost from the unacylated glycerol moiety of phosphatidylglycerol. Also, lipids other that phosphatidylglycerol, except diacylglycerol, and glycerol and glycerophosphate incorporated much less radioactivity. The [32P]phosphoryl group was also transferred from phosphatidylglycerol to lipoteichoic acid almost quantitatively in B. subtilis W23. A unique metabolism of phosphatidylglycerol was found in Bacillus sp. strain A007 which lacked phosphoglycolipid and lipoteichoic acid, that is, the turnover of phosphatidylglycerol of this organism was less extensive compared with that of B. subtilis W23, and both glycerol moieties of the lipid were metabolized at an identical rate. These results suggested that the major reaction involved in the turnover of phosphatidylglycerol was the transfer of glycerophosphate residue to lipoteichoic acid in a bacterium which possessed lipoteichoic acid and that several minor reactions also were involved in phosphatidylglycerol turnover.     

Competitive inhibition of lipolytic enzymes. VI. Inhibition of two human phospholipases A2 by acylamino phospholipid analogues.

The competitive inhibition of human pancreatic and a mutant human platelet phospholipase A2 (PLA2) was investigated using acylamino phospholipid analogues, which are potent competitive inhibitors of porcine pancreatic PLA2 [De Haas et al. (1990) Biochim. Biophys. Acta 1046, 249-257]. Both the mutant platelet PLA2 and the human pancreatic PLA2 are effectively inhibited by these compounds. The enzyme from platelets is most strongly inhibited by compounds with a negatively charged phosphoglycol headgroup. Compounds with a neutral phosphocholine headgroup are only weak inhibitors, whereas an inhibitor with a phosphoethanolamine headgroup shows an intermediate inhibitory capacity. The platelet PLA2 is most effectively inhibited by negatively charged inhibitors having a relatively short (four or more carbon atoms) alkylchain on position one and a acylamino chain of 14 carbon atoms on position two. For the pancreatic enzyme an inhibitor with a phosphoethanolamine headgroup was more effective than inhibitors with either a phosphocholine or a phosphoglycol headgroup. The chainlength preference of the pancreatic enzyme resembles that of the platelet PLA2. The largest discrimination in inhibition between the human platelet and the human pancreatic PLA2 is obtained with inhibitors with a negatively charged phosphoglycol headgroup, an alkyl chain of four carbon atoms on position one and a long acylamino chain of 14-16 carbon atoms on position two. Because the platelet PLA2 is thought to have several biological functions, specific inhibitors of this enzyme could have important implications in the design of pharmaceutically interesting compounds.     

Electric charge effects on phospholipid headgroups. Phosphatidylcholine in mixtures with cationic and anionic amphiphiles

The influence of electric surface charges on the polar headgroups and the hydrocarbon region of phospholipid membranes was studied by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) with charged amphiphiles. A positive surface charge was generated with dialkyldimethylammonium salts and a negative surface charge with dialkyl phosphates. The POPC:amphiphile ratio and hence the surface charge density could be varied over a large range since stable liquid-crystalline bilayers were obtained even for the pure amphiphiles in water. POPC was selectively deuterated at both methylene segments of the choline moiety and at the cis double bond of the oleic acyl chain. Additional experiments were carried out with 1,2-dipalmitoyl-rac-glycero-3-phosphocholine labeled at the C-2 position of the glycerol backbone. Deuterium, phosphorus, and nitrogen-14 nuclear magnetic resonance (NMR) spectra were recorded for liquid-crystalline bilayers with varying concentrations of amphiphiles. Although the hydrocarbon region and the glycerol backbone were not significantly influenced by the addition of amphiphiles, very large perturbations of the phosphocholine headgroup were observed. Qualitatively, these results were similar to those observed previously with other cationic and anionic molecules and suggest that the electric surface charge is the essential driving force in changing the phospholipid headgroup orientation and conformation. While the P-N dipole is approximately parallel to the membrane surface in the pure phospholipid membrane, the addition of a positively charged amphiphile or the binding of cationic molecules moves the N+ end of the dipole toward the water phase, changing the orientation of the phosphate segment by more than 30 degrees at the highest amphiphile concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis of charged amphipathic nitroxide lipid spin labels and an example of their application in membrane studies

The synthesis of a series of amphipathic nitroxide lipid spin labels is reported. Thus, 12-proxylhexadecanol has been converted into the versatile fatty acid spin label 14-proxylstearic acid. This substance was used to prepare 14-proxylstearyltrimethylammonium methanesulfonate, a positively charged label, and 14-proxylstearylmethyl phosphate sodium salt, a negatively charged label. Also prepared in the doxyl series were quaternary ammonium salts derived from 16-doxyl- and 7-doxylstearic acid. The positively charged and negatively charged proxyl labels were used in a preliminary experiment to investigate the role of charge in their interaction with reconstituted cytochrome oxidase. The average binding affinity of the negatively charged label is approximately 2-fold higher than that of the positively charged label at pH 7.4. At pH 5.5 the average relative affinity for negatively charged label is about 3.5-fold higher than that of positively charged label, suggesting that the ionizable group(s) on the protein can interact with the lipid headgroup.     

Effects of temperature and host cell genetic characteristics on the replication of the lipid-containing bacteriophage PR4 in Escherichia coli.

The lipid-containing bacteriophage PR4 is of special intest because it can replicate in various gram-negative bacteria, including Escherichia coli, that carry one of a group of drug resistance plasmids. PR4 grown in E. coli strain PS2R contains about 10% lipid by weight, with the negatively charged phospholipid phosphatidylglycerol being the most abundant lipid in the virion. We now report the following. (i) PR4 attaches to E. coli with an attachment rate constant of Ka approximately 6.2 X 10(-10) ml/min, which is about twice that of the enveloped phage phi6 (to Pseudomonas phaseolicola), but a factor of 5 less than that of phage PM2 (to Pseudomonas BAL-31). (ii) Use of an E. coli glycerol auxotroph indicated that a normal amount of PR4 replication occurs only if glycerol starvation (inhibition of all phospholipid synthesis) begins no earlier than about halfway through the lytic cycle. (iii) Use of an E. coli fatty acid synthesis temperature-sensitive mutant and an E. coli phosphatidylethanolamine synthesis temperature-sensitive mutant indicate that PR4 replication can occur in the absence of either normal fatty acid synthesis or normal phospholipid synthesis if the infection takes place prior to the termination of overall cell growth and the onset of cell death, (iv) Whereas PR4 burst size in nutrient media at 30 degrees C to 42%C is about 40, the burst size at 20 degrees C is less than 3, Temperature-shift experiments show that the temperature late in infection determines the burst size.     

Sterically stabilized liposomes. Reduction in electrophoretic mobility but not electrostatic surface potential.

The electrophoretic mobility of liposomes containing a negatively charged derivative of phosphatidylethanolamine with a large headgroup composed of the hydrophilic polymer polyethylene glycol (PEG-PE) was determined by Doppler electrophoretic light scattering. The results show that this method is improved by the use of measurements at multiple angles to eliminate artifacts and that very small mobilities can be measured. The electrophoretic mobility of liposomes with 5 to 10 mol% PEG-PE is approximately -0.5 mu ms-1/Vcm-1 regardless of PEG-PE content compared with approximately -2 mu ms-1/Vcm-1 for similar liposomes but containing 7.5% phosphatidylglycerol (PG) instead of PEG-PE. Measurements of surface potential by distribution of an anionic fluorescent probe show that the PEG-PE imparts a negative charge identical to that by PG, consistent with the expectation of similar locations of the ionized phosphate responsible for the charge. The reduced mobility imparted by the surface bound PEG is attributed to a mechanism similar to that described for colloidal steric stabilization: hydrodynamic drag moves the hydrodynamic plane of shear, or the hydrodynamic radius, away from the charge-bearing plane, that of the phosphate moities. An extended length of approximately 50 A for the 2,000 molecular weight PEG is estimated from the reduction in electrophoretic mobility.     

Analogous recognition of phospholipids by insect phagocytes and mammalian macrophages

Phagocytic cells from larvae of the moth Heliothis virescens and peritoneal macrophages of mice were observed to preferentially bind negatively charged phospholipid vesicles containing phosphatidylglycerol or phosphatidylserine as compared to neutral or positively charged vesicles. Since phagocytes have retained their primitive function of endocytosis throughout evolution, the recognition of negatively charged phospholipids may be a primitive mechanism for the identification of potential targets by macrophages.     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

A (2)H NMR study of macroscopically aligned bilayer membranes containing interfacial hydroxyl residues

The polar interface of membranes containing phosphatidylglycerol or cholesterol was studied by (2)H nuclear magnetic resonance (NMR) as a function of membrane hydration. The membranes were macroscopically aligned and hydrated with deuterium oxide. Water uptake and membrane annealing was achieved under NMR control, using a novel hydration technique. Well-resolved (2)H quadrupolar doublets were obtained from individual hydroxyl residues and from the interlamellar water. The response of the phosphatidylglycerol headgroup and of the cholesterol molecule to the spontaneous evaporation of interlamellar water could be thus monitored continuously. It is shown that the phosphatidylglycerol headgroup undergoes changes of conformation and average orientation with respect to the membrane surface and that the off-axis motion of the cholesterol molecule decreases. The deuteron exchange between hydroxyl residues and surface-associated D(2)O was determined by an inversion transfer technique. The exchange rates of the hydroxyl residues in the phosphatidylglycerol headgroup were different and depended strongly on the total hydration of the membrane. Significantly lower and almost hydration-independent rates were obtained for cholesterol. These results will be discussed with reference to earlier reports on the headgroup dynamics of phosphatidylglycerol and on the interaction of cholesterol with the membrane-water interface.