Surface potential effects on metal ion binding to phosphatidylcholine membranes. 31P NMR study of lanthanide and calcium ion binding to egg-yolk lecithin vesicles

³¹P NMR of phosphatidylcholine (lecithin) from egg-yolk in sonicated vesicles has been measured in the presence of various ions. Addition of Ln³⁺³ or Ca²⁺ shifted the ³¹P resonance of the phosphate groups of the outer surface of the vesicles. These shifts were measured at varied lanthanide or Ca²⁺ concentration at different ionic strengths obtained by addition of NaCl. The shifts induced by Tb³⁺ and Ca²⁺ have been analyzed using the theory of the diffuse double layer. Corrections were introduced for the effect of the ionic strength on the activities of the ions. The binding efficiency is shown to be controlled by the electrostatic potential produced by the bound cations at the membrane surface. This potential is slightly modified due to weak chloride binding. Binding constants have been derived.     

The interaction of various lanthanide ions and some anions with phosphatidylcholine vesicle membranes. A 31P NMR study of the surface potential effects

The interaction of various lanthanide ions with vesicles of phosphatidylcholine from egg yolk has been followed by 31P NMR at 30 degrees C. From known magnetic properties of these ions, separation of the paramagnetic shift into a pure contact and a pseudo-contact part was carried out. Binding curves for the contact contribution (F curves) were obtained from vesicles in solutions of sodium salts with monovalent anions over a wide concentration range. These curves should be insensitive to any conformational effects due to ion binding. Indication of a conformational change in the lipid head group at low ion binding was obtained by studying the ratio between the contact and the pseudo-contact contributions. Besides the adsorption of lanthanide ions, specific anion binding to the surface was introduced to account for the enhanced chemical shifts (Cl- < Br- < NO3-). The results were analyzed in terms of the theory for the diffuse double layer (Gouy-Chapman-Grahame) with equilibrium conditions for the adsorbing cations and anions. Simulations of the titration curves furnished parameters for the ion-lipid interactions. The synergism between the cations and anions follows from the potential effects. Comparison of results with lanthanide ions and Ca2+ indicates that the anion adsorption probably depends on the nature of the adsorbed cation. Lanthanide ion binding to L-glycerophosphorylcholine is not influenced by sodium salts. The binding constant for this complex is weaker than with phosphatidylcholine. The chemical shifts for the lanthanide ion complexes with these two phosphorus compounds seem to be about the same.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: 27Al and 31P NMR spectroscopic studies

27Al and 31P nuclear magnetic resonance (NMR) spectroscopies were used to investigate aluminum interactions at pH 3.4 with model membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). A solution state 27Al NMR difference assay was developed to quantify aluminum binding to POPC multilamellar vesicles (MLVs). Corresponding one-dimensional (1D) fast magic angle spinning (MAS) 31P NMR spectra showed that aluminum induced the appearance of two new isotropic resonances for POPC shifted to -6.4 ppm and -9.6 ppm upfield relative to, and in slow exchange with, the control resonance at -0.6 ppm. Correlation of the (27)Al and (31)P NMR binding data revealed a 1:2 aluminum:phospholipid stoichiometry in the aluminum-bound complex at -9.6 ppm and a 1:1 aluminum:phospholipid stoichiometry in that at -6.4 ppm. Slow MAS 31P NMR spectra demonstrated shifts in the anisotropic chemical shift tensor components of the aluminum-bound POPC consistent with a close coordination of aluminum with phosphorus. A model of the aluminum-bis-phospholipid complex is proposed on the basis of these findings.     

Aluminum binding to phosphatidylcholine lipid bilayer membranes: aluminum exchange lifetimes from 31P NMR spectroscopy

Two-dimensional (2D) (31)P magic angle spinning (MAS) nuclear magnetic resonance (NMR) exchange spectroscopy (EXSY) demonstrated that aluminum binds to the phosphate group of phosphatidylcholine (PC) in multilamellar vesicles at pH 3.2, forming preferentially 2/1, in addition to 1/1 (PC/Al) complexes in slow exchange with one another, and with free PC, on the NMR timescale. Exchange rate constants between these three co-existing species were measured as a function of temperature using one-dimensional (1D) selective inversion recovery (SIR) (31)P MAS NMR. Over the temperature range from 5 to 35 degrees C all three exchange rate constants increased by roughly an order of magnitude from k approximately 1-2 to 10-14s(-1), exhibiting Arrhenius behavior with activation energies on the order of 30-45 kJ mol(-1) and correspondingly positive enthalpies of activation. Entropies of activation were uniformly negative, consistent with an ordered transition state. From a biological perspective, the results demonstrate that aluminum binding to PC in biomembranes is transient on a biologically relevant time scale, so that the lipid bilayer portion of biomembranes is unlikely to act as a long term repository for aluminum, but rather should be viewed as a temporary reservoir of biologically available aluminum.     

A 31P NMR study of the thermal transition of dipalmitoyl lecithin vesicles.

Phosphorous-31 nuclear magnetic resonance (31P NMR) was used to study the liquid crystalline transition of sonicated dipalmitoyl lecithin vesicles in D2O. Linewidths were dependent upon temperature and changed dramatically through the transition region. The potential usefulness of 31P NMR spectroscopy for probing the thermal behavior of phospholipid membranes is evaluated and discussed.     

2H and 31P NMR study of the interaction of general anesthetics with phosphatidylcholine membranes

The effect of the general anesthetics alpha-chloralose and chloral hydrate as well as the nonanesthetic beta-chloralose on the order of phosphatidylcholine and phosphatidylcholine-cholesterol liposomes has been examined by 2H nuclear magnetic resonance. Chloral hydrate interacts with the hydrophilic head-group region, causing a change in the torsion angle of the C alpha-C beta bond. The membrane interior is also disordered by the presence of this agent. alpha-Chloralose, on the other hand, disorders only the central position of the membrane. beta-Chloralose produces little significant change in bilayer order.     

Clustering of lecithin molecules in phosphatidylserine membranes induced by calcium ion binding to phosphatidylserine

The effects of calcium ion on phosphatidyl L-serine (PS) have been studied with PS membranes containing a lecithin spin label (L¹). The calcium ion makes the ESR spectra of the L¹ in PS membranes broadened owing to the intermolecular spin-spin exchange interactions. The results indicate that the calcium ion binds to PS molecules to form rapidly rigid calcium ion-bound PS aggregates, the lecithin molecules being thereby separated from the host PS bilayers to form clusters. The magnesium ion is ineffective for the aggregation and exerts a quite different effect only at much higher concentrations.     

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.     

15N and 31P solid-state NMR investigations on the orientation of zervamicin II and alamethicin in phosphatidylcholine membranes

The topologies of zervamicin II and alamethicin, labeled with (15)N uniformly, selectively, or specifically, have been investigated by oriented proton-decoupled (15)N solid-state NMR spectroscopy. Whereas at lipid-to-peptide (L/P) ratios of 50 (wt/wt) zervamicin II exhibits transmembrane alignments in 1,2-dicapryl (di-C10:0-PC) and 1,2-dilauroyl (di-C12:0-PC) phosphatidylcholine bilayers, it adopts orientations predominantly parallel to the membrane surface when the lengths of the fatty acyl chains are extended. The orientational order of zervamicin II increases with higher phospholipid concentrations, and considerable line narrowing is obtained in di-C10:0-PC/zervamicin II membranes at L/P ratios of 100 (wt/wt). In contrast to zervamicin, alamethicin is transmembrane throughout most, if not all, of its length when reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers. The (31)P solid-state NMR spectra of all phospholipid/peptaibol samples investigated show a high degree of headgroup order, indicating that the peptides do not distort the bilayer structure. The observed differences in peptide orientation between zervamicin and alamethicin are discussed with reference to differences in their lengths, helical conformations, distribution of (hydroxy)proline residues, and hydrophobic moments. Possible implications for peptaibol voltage-gating are also described.     

Interaction of alamethicin with lecithin bilayers: a 31P and 2H NMR study

The interaction of alamethicin with artificial lecithin multilamellar dispersions was investigated by nuclear magnetic resonance (NMR) and Raman spectroscopies. 31P NMR studies revealed perturbation of the lipid head groups in the presence of the icosapeptide. Simulation of the 31P NMR spectra indicated that the observed spectral changes could be attributed to slight variations in the average tilt angle of the head groups. In contrast, no noticeable effect of the peptide on the segmental order of the hydrophobic acyl chains of the lipid molecules was detected by 2H NMR and Raman spectroscopic measurements. Taken together, these results indicated that, in the absence of a transmembrane electric potential, alamethicin interacts primarily at the water-lipid interface without significant insertion or incorporation into the bilayer leaflet.     

Metal ion binding properties of hen ovalbumin and S-ovalbumin: characterization of the metal ion binding site by 31P NMR and water proton relaxation rate enhancements

In this study, water proton relaxation rate (PRR) enhancements have been used to characterize the binding of metal ions to native ovalbumin, ovalbumin in which phosphate has been enzymatically cleaved from one or both of the two protein phosphoserines, and a heat-stabilized form of the protein (S-ovalbumin). With Scatchard plots constructed from water PRR enhancements, it was found that native ovalbumin and S-ovalbumin had one strong binding site for Mn2+ ion (KD approximately equal to 6.0 X 10(-4) M). Alkaline phosphatase treated ovalbumin, a protein having a single phosphoserine, had one Mn2+ binding site of slightly weaker affinity (KD approximately equal to 8.3 X 10(-4) M), while acid phosphatase treated ovalbumin, a dephosphorylated protein, had two much weaker Mn2+ ion binding sites (KD approximately equal to 1.3 X 10(-3) M). Competitive binding studies on the native protein suggested that Zn2+ ion competes with Mn2+ for the single strong-affinity site (KD approximately equal to 6.1 X 10(-3) M) while Mg2+ and Ca2+ do not. In a second set of experiments, the paramagnetic contribution to the 31P spin-lattice (T1P) and spin-spin (T2P) relaxation times at three separate magnetic field strengths was measured. Correlation times tau c characterizing Mn2+-31P dipolar relaxation were estimated from the ratios of T1P/T2P at a single field and from the ratios of spin-lattice relaxation rates at three different field strengths. The correlation times so obtained, ranging from about 0.7 to 7.7 ns at the three field strengths, were used in calculating distances from the bound Mn2+ ion to the phosphoserines of native ovalbumin, S-ovalbumin, and alkaline phosphatase treated ovalbumins. It was determined that the phosphate of phosphoserine-68 was 5.95 +/- 0.26 and 6.29 +/- 0.18 A from the Mn2+ in the native and alkaline phosphatase treated protein, respectively, and 6.99 +/- 0.30 A away from the Mn2+ in S-ovalbumin. The phosphate of phosphoserine-344 was determined to be 5.31 +/- 0.20 and 5.75 +/- 0.10 A from the Mn2+ ion in native ovalbumin and S-ovalbumin, respectively. The 13C nucleus of [1-13C]galactose enzymatically transferred to the nonreducing end of the ovalbumin oligosaccharide chain was not found to be significantly relaxed by Mn2+ bound to the protein, even at 1:1 stoichiometric ratio of metal:protein. Using this, we estimate the nonreducing terminal of the ovalbumin oligosaccharide to be at least 39 A from the metal ion binding site on the protein.     

On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study

113Cd and 31P NMR have been used to investigate the interactions of inhibitors with the metal ion of bovine carboxypeptidase A, using 113Cd as a replacement for the native zinc atom. In the absence of inhibitor and over the pH range 6-9, no 113Cd resonance is visible at room temperature. Upon lowering the temperature to 270 K, however, a broad resonance can be seen at 120 ppm. These results are discussed in terms of possible sources for this resonance modulation. Binding of low molecular weight inhibitors containing potential metal-coordinating moieties results in the appearance of a sharp 113Cd resonance. These inhibitors all bind to the metal ion, a fact which is reflected in the chemical shift of the cadmium resonance and, for L-phenylalanine phosphoramidate phenyl ester, by two-bond 113Cd-31P spin-spin coupling of 30 Hz in the 31P resonance of the bound inhibitor. For inhibitors that coordinate to the metal ion via oxygen, the 113Cd chemical shift is in the range 127-137 ppm, whereas for sulfur coordination there is a downfield shift of approximately 210 ppm. The complexes of 113Cd-substituted carboxypeptidase A with the D and L isomers of thiolactic acid are distinguished by a difference of 11 ppm in the chemical shift of their cadmium resonances. The enzyme complex formed with the macromolecular inhibitor from potatoes, which fills the S1 and S2 subsites, shows one or possibly two closely spaced broad 113Cd resonances. Both the chemical shift and the line width of the 113Cd resonances of the [113Cd]carboxypeptidase-inhibitor complexes give valuable structural and dynamic information about the enzyme active site.     

2H and 31P NMR study of pentalysine interaction with headgroup deuterated phosphatidylcholine and phosphatidylserine

The interaction of cationic pentalysine with phospholipid membranes was studied by using phosphorus and deuterium Nuclear Magnetic Resonance (NMR) of headgroup deuterated dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylserine (DMPS). In the absence of pentalysine, some of the deuterium and phosphorus spectra of DMPC/DMPS 5:1 (m:m) membranes gave lineshapes similar to those of partially-oriented bilayers with the planes of the bilayers being parallel to the magnetic field. The deuterium NMR data show that the quadrupolar splittings of the deuterated methylenes of the DMPC headgroup are not affected by adsorption of pentalysine on the PC/PS membranes. By contrast, the pentalysine produces significant changes in the quadrupolar splittings of the negatively charged DMPS headgroup. The results are discussed in relation to previous ²H NMR investigations of phospholipid headgroup perturbations arising from bilayer interaction with cationic molecules.Abbreviations NMR nuclear magnetic resonance - DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine - DMPS 1,2-dimyristoyl-sn-glycero-3-phosphoserine - POPC 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine - POPG 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoglycerol - PC phosphatidylcholine - PS phosphatidyl serine - PG phosphatidylglycerol - HEPES N-(2-hydroxy-ethyl)piperazine-N-2-ethanesulfonic acid - TRIS tris-(hydroxymethyl)aminoethane - EDTA ethylenediamine-tetra-acetic acid     

Effects of diacylglycerols on the structure of phosphatidylcholine bilayers: a 2H and 31P NMR study

The interaction of four diacylglycerols (DAGs) with multilamellar phospholipid bilayers consisting either of dipalmitoylphosphatidylcholine (DPPC) or of a mixture of DPPC and bovine liver phosphatidylcholine (BL-PC) extracts was investigated by a combination of 31P and 2H NMR spectrometry. We found that saturated and unsaturated long-chain DAGs induce different types of perturbations into the bilayer structure. The saturated DAGs dipalmitin and distearin induce lateral phase separation of the lipids into (i) DAG-enriched gellike domains and (ii) relatively DAG-free regions in the liquid-crystalline phase. In the latter regions, the order parameters along the fatty acyl chains of DPPC are practically identical with the control. This phase separation effect was observed in both model systems studied, and its extent is dependent upon DAG concentration and temperature. Only bilayer phases were present upon addition of dipalmitin or distearin at all concentrations and temperatures studied. The unsaturated DAGs diolein and DAG derived from egg PC (egg-DAG) affect PC bilayers in the following two ways: (i) by increasing the order parameters of the side chains, as observed for both DPPC and BL-PC model systems; (ii) by inducing nonbilayer lipid phases, as observed for BL-PC, but not DPPC. At a concentration of 25 mol % of an unsaturated DAG in mixed PC bilayers, a peak corresponding to isotropic lipid conformation appeared and increased in intensity with increase in temperature, while at 32 mol % hexagonal and bilayer phases coexisted.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluoride effects on 31P NMR spectra of macrophages

31P High resolution nuclear magnetic resonance studies have been carried out on the P388D1 tumoral cell line and the BCG elicited alveolar rabbit macrophages both in sedimented cells and in perfused agarose-embedded cells. When the cells were sufficiently oxygenated, the phosphorylated sugars and ATP concentrations attained high levels. The intensity of the peak representing phosphorylated sugars varied inversely with ATP level when macrophagic cells were treated by NaF. The identities of the phosphorylated sugars were revealed by 1H and 31P NMR studies of the P 388D1 cells perchloric extracts.     

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.     

Effect of calcium on the dynamic behavior of sialylglycerolipids and phospholipids in mixed model membranes. A 2H and 31P NMR study

DTSL, a sialic acid bearing glyceroglycolipid, has been deuteriated at the C3 position of the sialic acid headgroup and at the C3 position of the glycerol backbone. The glycolipid was studied as a neat dispersion and in multilamellar dispersions of DMPC (at a concentration of 5-10 mol % relative to phospholipid), using 2H and 31P NMR. The quadrupolar splittings, delta v Q, of the headgroup deuterons were found to differ in the neat and mixed dispersion, suggesting different headgroup orientations in the two systems. In DTSL-DMPC liposomes, two quadrupolar splittings were observed, indicating that the axial and equatorial deuterons make different angles with respect to the axis of motional averaging. The splittings originating from the equatorial and axial deuterons were found to increase and decrease with increasing temperature, respectively, indicating a temperature-dependent change in average headgroup orientation. Longitudinal relaxation times, T1Z, were found to be short (3-6 ms). The field dependence of T1Z suggests that more than one motion governs relaxation. At 30.7 MHz a T1Z minimum was observed at approximately 40 degrees C. At 46.1 MHz the T1Z values were longer and increased with temperature, demonstrating that the dominant rigid-body motions of the headgroup at this field are in the rapid motional regime (greater than 10(8) s-1). DTSL labeled at the glycerol C3 position was studied in DMPC multilamellar dispersions. Whereas two quadrupolar splittings have been observed for other glycolipids labeled at this position, only a single delta nu Q was observed. This shows that the orientation of the C2-C3 segment of DTSL relative to the bilayer normal differs from that of other glycolipids.(ABSTRACT TRUNCATED AT 250 WORDS)