Orientation and flexibility of the choline head group in phosphatidylcholine bilayers

The average orientation and flexibility of the phosphorylcholine group are deduced from deuterium and phosphorus-31 nuclear magnetic resonance measurements of unsonicated phosphatidylcholine bilayers in the liquid crystalline state. The experimental data are consistent with a model in which the polar head group exhibits a restricted flexibility characterized by rapid transitions between two enantiomeric conformations. A completely flexible or a completely rigid head group structure can be excluded. The phosphorylcholine residue is found to be bent at the position of the phosphate group, due to a gauchegauche conformation of the phosphodiester linkage. The choline dipole is aligned parallel to the plane of the membrane, which is in agreement with X-ray and neutron diffraction studies. The average orientation of the phosphorylcholine group is therefore the same as that of the phosphorylethanolamine head group.     

Dielectric properties of the polar head group region of zwitterionic lipid bilayers.

A theoretical model describing the dielectric properties of the lipid membrane-water interface region was developed. The rotating polar head groups (e.g. phosphatidylcholine) were simulated as a collection of interacting dipoles imbedded in a nonhomogeneous dielectric. The interactions between the nearest neighborhood were explicitly taken into account, while the other interactions were evaluated by means of the continuum theories. The values of the dielectric constant, its anisotropy and the spontaneous polarization of the interface were evaluated. As an application, we calculated the energy of interaction between an ion and the membrane polar head group region. The results indicate a small spontaneous polarization of the interface (1-1.7 Debyes per lipid molecule) due to the tilting angle of the choline residue with respect to the membrane surface. This dipolar field partially compensates that of opposite orientation originating from the ester group region, giving calculated overall dipolar potentials in better agreement with the experimental data. Our model suggests also a very strong dielectric anisotropy of the interface region, the component of the dielectric constant perpendicular to the membrane plane being much smaller than the parallel component.     

Transbilayer phosphatidylcholine distributions in small unilamellar sphingomyelin-phosphatidylcholine vesicles: effect of altered polar head group

The effect of the altered polar head group of phosphatidylcholine (PC) on its transbilayer distributions in small unilamellar vesicles containing sphingomyelin (SM) was ascertained with phospholipase A2 as the external membrane probe. These vesicles were formed by sonication and fractionated by centrifugation. The vesicle size was determined by gel-permeation chromatography and solute entrapment. Experiments were done to confirm that phospholipase A2 treatments did not induce fusion, lyse the vesicles, or cause PC to migrate across the vesicle bilayer. The complete degradation of external PC in intact vesicles was assured by carrying out the enzyme reactions in the absence as well as in the presence of 9.2 X 10(-5) M bovine serum albumin. In small vesicles comprised of SM and 30 mol % 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), DPPC preferentially distributed in the inner monolayer. This preference of DPPC in these vesicles disappeared upon introducing one C2H5 group at the carbon atom adjacent to the quaternary ammonium residue in its polar head group and was reversed when the C2H5 group was replaced by C6H5 and C6H5CH2 substituents or when the P-N distance was increased. These results indicate that the effective polar head-group volume is an important factor in determining the phospholipid distributions across the small vesicle bilayer.     

The conformation of the polar group of lysophosphatidylcholine in H2O: conformational changes induced by polyvalent cations

The conformation of the polar group of egg lysophosphatidylcholine and 1-myristoyl-sn-glycer-3-phosphorylcholine present as micelles in aqueous solution has been studied using NMR methods. In the absence of polyvalent cations the preferred conformation derived from spin-spin coupling constants is similar, but not identical, to that of phosphatidylethanolamine in the crystal structure (cf. Hitchcock, P.B., Mason, R., Thomas, K.M. and Shipley, G.F. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3036--3040). The presence of lanthanides induces a conformational change involving primarily the phosphorylcholine group, e.g. torsion angle alpha5 changes from an all gauche to an approximate trans disposition. The gauche leads to trans transitions observed with torsion angles alpha3 and alpha5 produce a more extended orientation of the polar group (relative to the hydrocarbon chain axis). In the presence of lanthanides the conformation of lysophosphatidylcholine is very similar to that of the diacyl phosphatidylcholines observed in fully hydrated bilayers (cf. Hauser, H., Phillips, M.C., Levine, B.A. and Williams, R.J.P. (1976) Nature 261, 390--394) with the P-N vector at an angle of about 45 degrees to the bilayer.     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.     

Phase separation in dipalmitoyl phosphatidylcholine bilayers induced by ionophores and binary electrolytes

Thermotropic behavior of unsonicated aqueous dispersion of dipalmitoyl phosphatidylcholine (DPPC) has been studied by scanning microcalorimetry and fluorescent probe method. Phase separation in the lipid bilayers was observed for systems containing ionophores (valinomycin, dinactin) and 1 : 1 electrolytes (NaCl, KCl, RbCl, CsCl). The ratio of lipid phases coexisting in the systems appeared to be dependent on the concentration of the electrolytes. Changes in the thermotropic properties of the lipid phase induced by valinomycin were observed when K+ and Rb+ ions-forming complexes with the ionophore were present in the systems. The latter phenomenon was not found for the systems containing dinactin possessing a lower ability for complex formation with the cations.     

Conformational difference in the polar groups of phosphatidylcholine and phosphatidylethanolamine in aqueous phase.

Proton and phosphorus nuclear magnetic resonance was used to investigate conformations of o-phosphorylcholine(OPC), o-phosphorylethanolamine(OPE) and L-alpha-glycerophosphorylethanolamine in aqueous solution, and the conformations of dipalmitoyl-3-sn-phosphatidylcholine and phosphatidylethanolamine from E. coli in methanol and chloroform solutions. It has been shown that in every case the O-C-C-N system prefers a gauche conformations, but in the choline moiety the dihedral angle around the C-C bond is distorted from the usual gauche angle, 60 degrees, to a larger one. The dihedral angle of OPC is shown to be more variable than that of OPE. This may be due to the curvature of its potential curve, i.e. asymmetrical curvature around the gauche minima. This property of the phosphatidylcholine molecule may be partly responsible for the flexibility of the phosphatidylcholine bilayer. The coupling is dominant in the P-O-C-C systems of the 5 compounds examined. The results also indicated that the two hydrocarbon chains in phosphatidylcholine or phosphatidylethanolamine are apt to take nearly parallel orientation in methanol solution. This characteristic is favourable for the formation of the bilayer structure.     

Conformational changes induced by binding of divalent cations to calregulin

Scatchard analysis of equilibrium dialysis studies have revealed that in the presence of 3.0 mM MgCl2 and 150 mM KCl, calregulin has a single binding site for Ca2+ with an apparent dissociation constant (apparent Kd) of 0.05 microM and 14 binding sites for Zn2+ with apparent Kd(Zn2+) of 310 microM. Ca2+ binding to calregulin induces a 5% increase in the intensity of intrinsic fluorescence and a 2-3-nm blue shift in emission maximum. Zn2+ binding to calregulin causes a dose-dependent increase of about 250% in its intrinsic fluorescence intensity and a red shift in the emission maximum of about 11 nm. Half-maximal wavelength shift occurs at 0.4 mol of Zn2+/mol of calregulin, and 100% of the wavelength shift is complete at 2 mol of Zn2+/mol of calregulin. In the presence of Zn2+ and calregulin the fluorescence intensity of the hydrophobic fluorescent probe 8-anilino-1-napthalenesulfonate (ANS) was enhanced 300-400% with a shift in emission maximum from 500 to 480 nm. Half-maximal Zn2+-induced shift in ANS emission maximum occurred at 1.2 mol of Zn2+/mol of calregulin, and 100% of this shift occurred at 6 mol of Zn2+/mol of calregulin. Of 12 cations tested, only Zn2+ and Ca2+ produced changes in calregulin intrinsic fluorescence, and none of these metal ions could inhibit the Zn2+-induced red shift in intrinsic fluorescence emission maximum. Furthermore, none of these cations could inhibit or mimic the Zn2+-induced blue shift in ANS emission maximum. These results suggest that calregulin contains distinct and specific ligand-binding sites for Ca2+ and Zn2+. While Ca2+ binding results in the movement of tryptophan away from the solvent, Zn2+ causes a movement of tryptophan into the solvent and the exposure of a domain with considerable hydrophobic character.     

Hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, induced permeability change of phosphatidylcholine bilayers

Interactions of hypelcin A, an alpha-aminoisobutyric acid containing antibiotic peptide, with phosphatidylcholine vesicles were investigated to obtain information on its bioactive mechanism. The peptide induced the leakage of a fluorescent dye, calcein, entrapped in sonicated vesicles. The leakage rate depended on both the peptide and the lipid concentrations. Analysis of this dependency indicated that the leakage was due to the monomeric peptide and that the membrane-perturbing activity of the monomer was higher for solid distearoylphosphatidylcholine vesicles than for fluid egg yolk phosphatidylcholine vesicles. Hypelcin A also affected the gel to liquid-crystalline phase transition of dipalmitoylphosphatidylcholine multilamellar vesicles. The transition was broadened with a reduced transition enthalpy, suggesting the peptide strongly binds the surrounding lipids to perturb the bilayer lipid packing. A circular dichroism study revealed that the helical content of hypelcin A increases upon membrane binding. We concluded that the monomeric peptide with an increased helical content, complexed with the lipids, perturbs the lipid organization and induces the increased permeability.     

Thiocyanate and bromide ions influence the bilayer structural parameters of phosphatidylcholine bilayers

The influence of monovalent cations and anions on the structural parameters of dipalmitoylphosphatidylcholine (DPPC) bilayers was examined at 25 degrees C using X-ray diffraction. It was shown that monovalent salts, in general, have little effect on lipid packing within the bilayer. However, fully hydrated DPPC bilayers in 1 M KSCN pack in an interdigitated acyl chain phase. This is the first observation of an ion-induced interdigitated bilayer phase in a zwitterionic lipid. In addition, gel state DPPC bilayers in 1 M KBr imbibe approx. 10 A more solvent than bilayers in water. The influence of these same salts on the phase transitions of DPPC bilayers was also examined using high-resolution differential scanning calorimetry. These results are discussed in terms of ion-induced changes in solvent and solvent/bilayer structure.     

Morphological changes of phosphatidylcholine bilayers induced by melittin: vesicularization, fusion, discoidal particles

Morphological changes induced by the melittin tetramer on bilayers of egg phosphatidylcholine and dipalmitoylphosphatidylcholine have been studied by quasi-elastic light scattering, gel filtration and freeze-fracture electron microscopy. It is concluded that melittin similarly binds and changes the morphology of both single and multilamellar vesicles, provided that their hydrocarbon chains have a disordered conformation, i.e., at temperatures higher than that of the transition, Tm. When the hydrocarbon chains are ordered (gel phase), only small unilamellar vesicles are morphologically affected by melittin. However after incubation at T greater than Tm, major structural changes are detected in the gel phase, regardless of the initial morphology of the lipids. Results from all techniques agree on the following points. At low melittin content, phospholipid-to-peptide molar ratios, Ri greater than 30, heterogeneous systems are observed, the new structures coexisting with the original ones. For lipids in the fluid phase and Ri greater than 12, the complexes formed are large unilamellar vesicles of about 1300 +/- 300 A diameter and showing on freeze-fracture images rough fracture surfaces. For lipids in the gel phase, T less than Tm after passage above Tm, and for 5 less than Ri less than 50, disc-like complexes are observed and isolated. They have a diameter of 235 +/- 23 A and are about one bilayer thick; their composition corresponds to one melittin for about 20 +/- 2 lipid molecules. It is proposed that the discs are constituted by about 1500 lipid molecules arranged in a bilayer and surrounded by a belt of melittin in which the mellitin rods are perpendicular to the bilayer. For high amounts of melittin, Ri less than 2, much smaller and more spherical objects are observed. They are interpreted as corresponding to lipid-peptide co-micelles in which probably no more bilayer structure is left. It is concluded that melittin induces a reorganization of lipid assemblies which can involve different processes, depending on experimental conditions: vesicularization of multibilayers; fusion of small lipid vesicles; fragmentation into discs and micelles. Such processes are discussed in connexion with the mechanism of action of melittin: the lysis of biological membranes and the synergism between melittin and phospholipases.     

Visualization of highly ordered striated domains induced by transmembrane peptides in supported phosphatidylcholine bilayers

We used atomic force microscopy (AFM) to study the lateral organization of transmembrane TmAW(2)(LA)(n)W(2)Etn peptides (WALP peptides) incorporated in phospholipid bilayers. These well-studied model peptides consist of a hydrophobic alanine-leucine stretch of variable length, flanked on each side by two tryptophans. They were incorporated in saturated phosphatidylcholine (PC) vesicles, which were deposited on a solid substrate via the vesicle fusion method, yielding hydrated gel-state supported bilayers. At low concentrations (1 mol %) WALP peptides induced primarily line-type depressions in the bilayer. In addition, striated lateral domains were observed, which increased in amount and size (from 25 nm up to 10 microm) upon increasing peptide concentration. At high peptide concentration (10 mol %), the bilayer consisted mainly of striated domains. The striated domains consist of line-type depressions and elevations with a repeat distance of 8 nm, which form an extremely ordered, predominantly hexagonal pattern. Overall, this pattern was independent of the length of the peptides (19-27 amino acids) and the length of the lipid acyl chains (16-18 carbon atoms). The striated domains could be pushed down reversibly by the AFM tip and are thermodynamically stable. This is the first direct visualization of alpha-helical transmembrane peptide-lipid domains in a bilayer. We propose that these striated domains consist of arrays of WALP peptides and fluidlike PC molecules, which appear as low lines. The presence of the peptides perturbs the bilayer organization, resulting in a decrease in the tilt of the lipids between the peptide arrays. These lipids therefore appear as high lines.     

Molecular organization in striated domains induced by transmembrane alpha-helical peptides in dipalmitoyl phosphatidylcholine bilayers

Transmembrane (TM) alpha-helical peptides with neutral flanking residues such as tryptophan form highly ordered striated domains when incorporated in gel-state 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) bilayers and inspected by atomic force microscopy (AFM) (1). In this study, we analyze the molecular organization of these striated domains using AFM, photo-cross-linking, fluorescence spectroscopy, nuclear magnetic resonance (NMR), and X-ray diffraction techniques on different functionalized TM peptides. The results demonstrate that the striated domains consist of linear arrays of single TM peptides with a dominantly antiparallel organization in which the peptides interact with each other and with lipids. The peptide arrays are regularly spaced by +/-8.5 nm and are separated by somewhat perturbed gel-state lipids with hexagonally organized acyl chains, which have lost their tilt. This system provides an example of how domains of peptides and lipids can be formed in membranes as a result of a combination of specific peptide-peptide and peptide-lipid interactions.     

The conformation of the polar group of lysophosphatidylcholine in H2O; conformational changes induced by polyvalent cations

The conformation of the polar group of egg lysophosphatidylcholine and 1-myristoyl-sn-glycero-3-phosphorylcholine present as micelles in aqueous solution has been studied using NMR methods. In the absence of polyvalent cations the preferred conformation derived from spin-spin coupling constants is similar, but not identical, to that of phosphatidylethanolamine in the crystal structure (cf. Hitchcock, P.B., Mason, R., Thomas, K.M. and Shipley, G.G. (1974) Proc. Natl. Acad. Sci. U.S. 71, 3036–3040). The presence of lanthanides induces a conformational change involving primarily the phosphorylcholine group, e.g. torsion angle $\text{α}{}_5$ changes from an all gauche to an approximate trans disposition. The gauche → trans transitions observed with torsion angles α₃ and α₅ produce a more extended orientation of the polar group (relative to the hydrocarbon chain axis). In the presence of lanthanides the conformation of lysophosphatidylcholine is very similar to that of the diacyl phosphatidylcholines observed in fully hydrated bilayers (cf. Hauser, H., Phillips, M.C., Levine, B.A. and Williams, R.J.P. (1976) Nature 261, 390–394) with the P-N vector at an angle of about 45° to the bilayer.     

Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations

Manganese ion, like Mg2+, has been found to produce high biosynthetic activity of the unadenylylated form of glutamine synthetase obtained from Mycobacterium smegmatis, and the activity with each of these cations was decreased by the adenylylation of the enzyme. Further, the gamma-glutamyltransferase reaction was catalyzed in the presence of either Mn2+, Mg2+, or Co2+ with both unadenylylated and adenylylated enzyme; however, each of these divalent cation-dependent activities was also decreased by one order of magnitude by adenylylation of the enzyme. From studies of UV-difference spectra, it was found that the ability of M. smegmatis glutamine synthetase to assume a number of distinctly different configurations was the result of the varied response of the enzyme to different cations. When either Mn2+, Mg2+, Ca2+, or Co2+ was added to the relaxed (divalent cation-free) enzyme at saturated concentration, each produced a similar UV-difference spectrum of the enzyme, indicating that the conformational states induced by these cations are similar with respect to the polarity of the microenvironment surrounding the tyrosyl and tryptophanyl groups of the enzyme. The binding of Cd2+, Ni2+, or Zn2+ to the relaxed enzyme each produced a different shift in the UV-absorption spectrum of the enzyme, indicating different conformational states. The kinetics of the spectral change that occurred upon addition of Mn2+, Mg2+, or Co2+ to a relaxed enzyme preparation were determined. The first-order rate constants for the decrease in relaxed enzyme with Mn2+ and Mg2+ were 0.604 min-1 and 0.399 min-1, respectively, at 25 degrees C, pH 7.4. The spectral change with Co2+ was completed within the time of mixing (less than 4 s). For these three metal ions, the total spectral change as well as the time course of the change were the same for both the unadenylylated enzyme and the partially adenylylated enzyme. However, Hill coefficients obtained from spectrophotometric titration data for both Mn2+ and Mg2+ were decreased with adenylylated enzyme to compared with unadenylylated enzyme. These results suggest that covalently bound AMP on each subunit may be involved in subunit interactions within the dodecamer. Circular dichroism measurements also indicated that the various structural changes of the M. smegmatis glutamine synthetase were produced by the binding of the divalent cations.     

Lateral distribution of a pyrene-labeled phosphatidylcholine in phosphatidylcholine bilayers: fluorescence phase and modulation study

The lateral distribution of 1-palmitoyl-2-[10-(1-pyrenyl)decanoyl]phosphatidylcholine (PyrPC) was studied in small unilamellar vesicles of 1,2-dipalmitoyl-, 1,2-dimyristoyl-, and 1-palmitoyl-2-oleoyl-phosphatidylcholine (DPPC, DMPC, and POPC, respectively) under anaerobic conditions. The DPPC and DMPC experiments were carried out over temperature ranges above and below the matrix phospholipid phase transition temperature (Tm). The excimer to monomer fluorescence intensity ratio (E/M) was determined as a function of temperature for the three PyrPC/lipid mixtures. Phase and modulation data were used to determine the temperature dependence of pyrene fluorescence rate parameters in gel and in liquid-crystalline bilayers. These parameters were then used to provide information about excited-state fluorescence in phospholipid bilayers, calculate the concentration of the probe within liquid-crystalline and gel domains in the phase transition region of PyrPC in DPPC, and simulate E/M vs. temperature curves for three systems whose phase diagrams are different. From the simulated curves we could determine the relationship between the shape of the three simulated E/M vs. temperature curves and the lateral distribution of the probe. This information was then used to interpret the three experimentally derived E/M vs. temperature curves. Our results indicate that PyrPC is randomly distributed in pure gel and fluid phosphatidylcholine bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Configuration and interactions of the polar head group in gangliosides

1. The interactions of gangliosides with Ca²⁺ and some polar-head-group requirements for establishment of particular interactions with phosphatidylcholine were studied in monolayers at the air/145mm-NaCl interface. 2. Ganglioside–Ca²⁺ interactions, as revealed by surface-potential measurements, depended on the position occupied by sialosyl residues in the oligosaccharide chain. The interactions with Ca²⁺ of the single sialosyl residue of monosialogangliosides occurred above 0.1mm-CaCl₂, whereas the interaction of the cation with additional sialosyl groups in di- or tri-sialogangliosides depended on the carbohydrate residue to which the sialosyl moiety was attached. The sialosyl residue bound in sialosyl–sialosyl linkage interacted very little with Ca²⁺. The sialosyl residue attached to the terminal galactose of the neutral tetrasaccharide chain interacted with Ca²⁺ above 1μm-CaCl₂. 3. Experiments with mixed monolayers containing dihexadecyl phosphate and hexadecyltrimethylammonium indicated that for the occurrence of interactions of polysialogangliosides with phosphatidylcholine characterized by reductions in molecular packing and surface potential both charged groups of the phospholipid and sialosyl residues with particular dipolar properties in the ganglioside are participating. 4. Possible configurations that can explain the behaviour in monolayers were inspected with space-filling molecular models. The position of the carboxylate group of sialosyl residues with respect to the interface and to the sialosyl molecular plane can explain the different orientation of the dipole-moment vector of this residue, which depends on the position to which it is linked in the oligosaccharide chain. Favoured interactions of polysialogangliosides with phosphatidylcholine may result from a configuration allowing a partial matching of two oppositely oriented electrical vectors contributed by the zwitterionic phosphocholine group and particular sialosyl groups.