Interaction of epicatechin gallate with phospholipid membranes as revealed by solid-state NMR spectroscopy

Epicatechin gallate (ECg), a green tea polyphenol, has various physiological effects. Our previous nuclear Overhauser effect spectroscopy (NOESY) study using solution NMR spectroscopy demonstrated that ECg strongly interacts with the surface of phospholipid bilayers. However, the dynamic behavior of ECg in the phospholipid bilayers has not been clarified, especially the dynamics and molecular arrangement of the galloyl moiety, which supposedly has an important interactive role. In this study, we synthesized [13C]-ECg, in which the carbonyl carbon of the galloyl moiety was labeled by 13C isotope, and analyzed it by solid-state NMR spectroscopy. Solid-state 31P NMR analysis indicated that ECg changes the gel-to-liquid-crystalline phase transition temperature of DMPC bilayers as well as the dynamics and mobility of the phospholipids. In the solid-state 13C NMR analysis under static conditions, the carbonyl carbon signal of the [13C]-ECg exhibited an axially symmetric powder pattern. This indicates that the ECg molecules rotate about an axis tilting at a constant angle to the bilayer normal. The accurate intermolecular-interatomic distance between the labeled carbonyl carbon of [13C]-ECg and the phosphorus of the phospholipid was determined to be 5.3±0.1 ? by 13C-(31)P rotational echo double resonance (REDOR) measurements. These results suggest that the galloyl moiety contributes to increasing the hydrophobicity of catechin molecules, and consequently to high affinity of galloyl-type catechins for phospholipid membranes, as well as to stabilization of catechin molecules in the phospholipid membranes by cation-π interaction between the galloyl ring and quaternary amine of the phospholipid head-group.     

The interaction of amino-deuteromethylated melittin with phospholipid membranes studied by deuterium NMR

Melittin, deuteromethylated on each of the four amino groups (Gly-1 N alpha and Lys-7, 21, and 23 N epsilon), was prepared by reductive methylation using deuteroformaldehyde and NaBD3CN. Deuterium NMR spectra were obtained for the modified peptide (D-melittin) bound to phospholipid bilayers and erythrocyte ghosts. D-Melittin at 4 mol% (peptide:lipid) induced reversible transitions between extended bilayers and micelles at the phase-transition temperature in dimyristoylphosphatidylcholine (DMPC) bilayers. These changes in lipid morphology did not occur at 1 mol% D-melittin: DMPC and the peptide was highly motionally restricted in gel in gel-phase lipid.     

Modulation of melittin-induced lysis by surface charge density of membranes.

Phosphorus NMR spectroscopy was used to characterize the importance of electrostatic interactions in the lytic activity of melittin, a cationic peptide. The micellization induced by melittin has been characterized for several lipid mixtures composed of saturated phosphatidylcholine (PC) and a limited amount of charged lipid. For these systems, the thermal polymorphism is similar to the one observed for pure PC: small comicelles are stable in the gel phase and extended bilayers are formed in the liquid crystalline phase. Vesicle surface charge density influences strongly the micellization. Our results show that the presence of negatively charged lipids (phospholipid or unprotonated fatty acid) reduces the proportion of lysed vesicles. Conversely, the presence of positively charged lipids leads to a promotion of the lytic activity of the peptide. The modulation of the lytic effect is proposed to originate from the electrostatic interactions between the peptide and the bilayer surface. Attractive interactions anchor the peptide at the surface and, as a consequence, inhibit its lytic activity. Conversely, repulsive interactions favor the redistribution of melittin into the bilayer, causing enhanced lysis. A quantitative analysis of the interaction between melittin and negatively charged bilayers suggests that electroneutrality is reached at the surface, before micellization. The surface charge density of the lipid layer appears to be a determining factor for the lipid/peptide stoichiometry of the comicelles; a decrease in the lipid/peptide stoichiometry in the presence of negatively charged lipids appears to be a general consequence of the higher affinity of melittin for these membranes.     

Interaction of the Saccharomyces cerevisiae alpha-factor with phospholipid vesicles as revealed by proton and phosphorus NMR

Proton and phosphorus-31 nuclear magnetic resonance (1H and 31P NMR) studies of the interaction between a tridecapeptide pheromone, the alpha-factor of Saccharomyces cerevisiae, and sonicated lipid vesicles are reported. 31P NMR studies demonstrate that there is interaction of the peptide with the phosphorus headgroups, and quasielastic light scattering (QLS) studies indicate that lipid vesicles increase in size upon addition of peptide. Previous solution (aqueous and DMSO) studies from this laboratory indicate that alpha-factor is highly flexible with only one long-lived identifiable structural feature, a type II beta-turn spanning the central portion of the peptide. Two-dimensional (2D) 1H nuclear Overhauser effect spectroscopy (NOESY) studies demonstrate a marked ordering of the peptide upon interaction with lipid, suggesting a compact N-terminus, in addition to a stabilized beta-turn. In contrast to our results in both solution and lipid environment, Wakamatsu et al. [Wakamatsu, K., Okada, A., Suzuki, M., Higashijima, T., Masui, Y., Sakakibara, S., & Miyazawa, T. (1986) Eur. J. Biochem. 154, 607-615] proposed a lipid environment conformation, on the basis of one-dimensional transferred NOE studies in D2O, which does not include the beta-turn.     

Partitioning behaviour of 1-hexanol into lipid membranes as studied by deuterium NMR spectroscopy

Deuterium nuclear magnetic resonance (NMR) spectroscopy was used to study the partitioning behaviour of 1-hexanol specifically deuterated in the alpha-position into model lipid bilayers. In all systems studied, the observed deuterium NMR lineshapes were time-dependent. Initially, 1-hexanol-d2 gave rise to an isotropic deuterium resonance with a different chemical shift from that of aqueous 1-hexanol-d2. After equilibration over a period of days, a broader spectral component characteristic of a spherically-averaged powder-pattern was observed. The quadrupole anisotropy of the 1-hexanol-d2 giving rise to the broad spectrum depended upon the cholesterol content of the membrane. From quantitation of the anisotropic to isotropic deuterium NMR spectra, the partition coefficients of 1-hexanol-d2 in a number of bilayer systems (asolectin and phosphatidylcholine bilayers (the latter with and without cholesterol] were determined. The partitioning of 1-hexanol-d2 into red blood cell membranes, and a suspension of lipids extracted from red blood cell membranes, was also examined. It is suggested that 1-hexanol, and probably other lipophiles, can partition to either the bilayer surface or the bilayer interior in a time-dependent manner.     

Phase fluctuation in phospholipid membranes revealed by Laurdan fluorescence.

The organization of lipids surrounding membrane proteins can influence their properties. We have used 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) to study phase coexistence and phase interconversion in membrane model systems. The fluorescence properties of Laurdan provide a unique possibility to study lipid domains because of the different excitation and emission spectra of this probe in the gel and in the liquid-crystalline phase. The difference in excitation spectra allows photoselection of Laurdan molecules in one of the two phases. Using the difference in emission spectra it is then possible to observe interconversion between the two phases. We have performed experiments in dipalmitoyl-phosphatidylcholine (DPPC) vesicles at different temperatures, in particular in the region of the phase transition, where phase coexistence and interconversion between phases is likely to be maximal. We have also studied vesicles of different lipids and mixtures dilauroyl-phosphatidylcholine (DLPC), DPPC, and 50% DLPC in DPPC. Both steady-state fluorescence intensity and polarization data have been collected. To quantitate phase coexistence and interconversion we have introduced the concept of "generalized polarization." We have also performed time-resolved experiments to directly prove the interconversion process. We have found that in DLPC-DPPC mixtures, at 20 degrees C, phase interconversion occurs in approximately 30-40 ns.     

Direct observation of the properties of cholesterol in membranes by deuterium NMR

The properties of cholesterol in bilayers of egg phosphatidylcholine (PC) were investigated directly by means of ²H-NMR of specifically-deuterated species (C3, C7, C26, C27). Quadrupole splittings were a measure of molecular ordering, and relaxation times T₁ and T_2e were indicators of rates of motion. The importance of the use of echoes for spectral acquisition is emphasised, particularly to obtain accurate values of the quadrupole splitting. In the case of overlapping powder patterns from two labelled positions, the use of the absolute value mode of spectral presentation is shown to yield reasonable estimates of the individual quadrupole splittings. Spectral properties were monitored as a function of cholesterol concentration and temperature. Increasing cholesterol concentration led to a high degree of ordering for the rigid ring system of cholesterol, approaching a molecular order parameter of 0.8 at 50 mol% cholesterol. The isopropyl methyl groups were in all cases less ordered anmore mobile than the ring system, but responded in a similar fashion to variable cholesterol concentration and temperature. The observation of a minimum in the temperature dependence of T₁ for cholesterol-7,7-d₂ led to a direct estimate of its correlation time for molecular motion, 3.5 × 10^?9 s rad^?1. This indicates that the overall rate of motion of cholesterol is considerably slower than that of the lipids in which it is located. The short T_2e values suggest that the motional spectrum of cholesterol is rich in low frequencies. The parallel temperature and cholesterol dependences of quadrupole splittings for different positions on the rigid ring system of cholesterol indicate that the position of the axis of motional averaging of the molecule is not changing, and is the same as that determined in an earlier study. It is emphasised that the steep temperature dependence and small quadrupole splittings for the chain isopropyl methyl groups of cholesterol do not necessarily indicate a high degree of disorder, but may be due to their axes of motional averaging lying at angles close to 54° with respect to the director of the ordered lipids.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

Molecular order in acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids

The first application of deuterium magnetic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37°C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.     

Study of water permeability through phospholipid vesicle membranes by O NMR

Vesicle suspensions of up to 5 % egg lecithin and 2.5 % cholesterol have been found to have no effect on the NMR relaxation times of ¹⁷O from water. Addition of 1–5 mM Mn²⁺ to an equimolar vesicle suspension of egg lecithin and cholesterol permitted resolution of the free induction decay into two exponential components, a fast one arising from the external water and a slow one arising from the intravesicular fluid. From the rates of relaxation the mean life time of the water molecules within the vesicles was calculated to be 1±0.1 ms at 22°C. The size of the vesicle was estimated from electron micrographs to be about 500 Å in diameter. These data yield an equilibrium water permeability, P_w, of about 8 μs⁻¹ for the vesicle membranes. From the temperature dependence of P_w an activation energy of 12±2 kcal/mol was obtained. The longitudinal relaxation time (T₁) of water within vesicles remained the same as in pure water.     

Phospholipid phase transitions as revealed by NMR

Aqueous dispersions of phospholipids can adopt a range of polymorphic phases which include bilayer and non-bilayer forms. Within the bilayer form, laterally separated phases may be induced as a result of surface electrostatic associations, thermotropic behaviour, lipid-protein interactions or because of molecular mismatch between chemically distinct phospholipids. Nuclear magnetic resonance (NMR) methods, designed to exploit the properties of either indigenous nuclei or isotopic labels introduced specifically into a phospholipid, can be used in some cases to describe the molecular properties and behaviour of phospholipids in both macroscopically distinct phases and in molecularly distinct phases within the same polymorphic state. If the molecular motion of phospholipids in co-existing phases is sufficiently different, NMR methods can, in principle, give estimates of the life-time of the phases and the rate of molecular exchange between the phases.     

Molecular order in Acholeplasma laidlawii membranes as determined by deuterium magnetic resonance of biosynthetically-incorporated specifically-labelled lipids.

The first application of deuterium magentic resonance of specifically labelled lipids to the study of a natural biological membrane is described. Palmitic acid labelled at the terminal methyl group with deuterium was incorporated biosynthetically into the lipids of the plasma membrane of Acholeplasma laidlawii. The deuterium nuclear magnetic resonance spectra contain quadrupole splittings which yield directly order parameters for this region of the membrane. Below the growth temperature (37 degrees C) the spectra are indicative of lipid in both gel and liquid crystalline states. Above this temperature they demonstrate the existence of an entirely liquid crystalline membrane whose order parameter decreases rapidly with increasing temperature. Comparison with egg phosphatidylcholine over the same temperature range shows a more rapid change in order with temperature for the A. laidlawii membranes.     

Sterol orientations in phosphatidylcholine liposomes as determined by deuterium NMR

Deuterium magnetic resonance spectra (55.26 MHz) of cholesterol-3 alpha-d1 and epicholesterol-3 beta-d1 in dipalmitoylglycerophosphocholine (DPPC) liposomes were measured as a function of sterol-to-phospholipid ratio below (24 degrees C) and above (60 degrees C) the phase transition temperature of DPPC. From the quadrupolar splittings delta vq, the molecular order parameters S describing the motions of the sterols in the bilayer were calculated, and the most probable angle of tilt alpha 0 of the molecular axis of the sterols relative to the bilayer normal was determined. We observed that the molecular axis of cholesterol in DPPC liposomes at both 24 and 60 degrees C is tilted at an angle of 16-19 degrees with the 3 beta-hydroxyl group projecting parallel to the bilayer normal into the aqueous interface. In contrast, at 24 degrees C, epicholesterol is aligned parallel (0 degrees) to the bilayer normal, placing the 3 alpha-hydroxyl group essentially perpendicular to the bilayer normal along the aqueous interface. At 60 degrees C, the average angle of epicholesterol (16-18 degrees) is similar to that of cholesterol, which can project the 3 alpha-hydroxyl group into the hydrophobic bilayer region. On the basis of the observed tilt angles of the two isomeric sterols in DPPC liposomes, a model is proposed that can rationalize the differential effects of cholesterol and epicholesterol on membrane properties.(ABSTRACT TRUNCATED AT 250 WORDS)     

The nature of bile salt micelles as studied by deuterium NMR

Deuterium NMR of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid was studied. Molecular sizes obtained from deuterium spin-lattice relaxation time (T₁) data of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in methanol and in water are in accordance with monomeric and tetrameric structures in the two media, respectively. The deuterium T₁ and intensity of 3α,12α-dihydroxy-7,7-dideutero-5β-cholanoic acid in aqueous solution at pH 8.0–8.8 were studied as functions of NaCl and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaCl, and that 3α, 12α-dihydroxy-7,7-dideutero-5β-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

Study of water permeability through phospholipid vesicle membranes by 17O NMR.

Vesicle suspensions of up to 5% egg lecithin and 2.5% cholesterol have been found to have no effect on the NMR relaxation times of 17O from water. Addition of 1-5 mM Mn2+ to an equimolar vesicle suspension of egg lecithin and cholesterol permitted resolution of the free induction decay into two exponential components, a fast one arising from the external water and a slow one arising from the intravesicular fluid. From the rates of relaxation the mean life time of the water molecules within the vesicles was calculated to be 1+/- 0.1 ms at 22 degrees C. The size of the vesicle was estimated from electron micrographs to be about 500 A in diameter. These data yield an equilibrium water permeability, Pw, of about 8 mus-1 for the vesicle membranes. From the temperature dependence of Pw an activation energy of 12+/-2 kcal/mol was obtained. The longitudinal relaxation time (T1) of water within vesicles remained the same as in pure water.     

Molecular organization of cholesterol in polyunsaturated phospholipid membranes: a solid state 2H NMR investigation.

We compared the molecular organization of equimolar [3alpha-2H1]cholesterol in 18:0-18:1PC (1-stearoyl-2-oleoylphosphatidylcholine), 18:0-22:6PC (1-stearoyl-2-docosahexaenoylphosphatidylcholine), 18:0-20:4PC (1-stearoyl-2-arachidonylphosphatidylcholine) and 20:4-20:4PC (1,2-diarachidonylphosphatidylcholine) bilayers by solid state 2H NMR. Essentially identical quadrupolar splittings (delta v(r) = 45 +/- 1 kHz) corresponding to the same molecular orientation characterized by tilt angle alpha0 = 16 +/- 1 degrees were measured in 18:0-18:1PC, 18:0-22:6PC and 18:0-20:4PC. A profound difference in molecular interaction with dipolyunsaturated 20:4-20:4PC, in contrast, is indicated for the sterol. Specifically, the tilt angle alpha0 = 22 +/- 1 degrees (derived from delta v(r) = 37 +/- 1 kHz) is greater and its membrane intercalation is only 15 mol%.     

The effect of ceramide on phosphatidylcholine membranes: a deuterium NMR study

Biological membranes contain domains having distinct physical properties. We study defined mixtures of phosphoglycerolipids and sphingolipids to ascertain the fundamental interactions governing these lipids in the absence of other cell membrane components. By using (2)H-NMR we have determined the temperature and composition dependencies of membrane structure and phase behavior for aqueous dispersions of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the ceramide (Cer) N-palmitoyl-sphingosine. It is found that gel and liquid-crystalline phases coexist over a wide range of temperature and composition. Domains of different composition and phase state are present in POPC/Cer membranes at physiological temperature for Cer concentrations exceeding 15 mol %. The acyl chains of liquid crystalline phase POPC are ordered by the presence of Cer. Moreover, Cer's chain ordering is greater than that of POPC in the liquid crystalline phase. However, there is no evidence of liquid-liquid phase separation in the liquid crystalline region of the POPC/Cer phase diagram.     

The nature of bile salt micelles as studied by deuterium NMR.

Deuterium NMR of 3alpha,12alpha-dihydroxy-7,7dideutero-5beta-cholanic acid was studied. Molcular sizes obtained from deuterium spin-lattice relaxation time (T1) data of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in methanol and in water are in accordance with monometic and tetrameric structures in the two media, respectively. The deuterium T1 and intensity of 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid in aqueous solution at pH 8.0--8.8 were studied as functions of NcC1 and lecithin concentrations. The results indicated that tetramers are in equilibrium with larger aggregates when secondary micelles are formed in the precense of NaC1, and that 3alpha,12alpha-dihydroxy-7,7-dideutero-5beta-cholanoic acid forms mixed micelles with lecithin with a molecular ratio of 2 : 3.     

Molecular view of hexagonal phase formation in phospholipid membranes

Important biological processes, such as vesicle fusion or budding, require the cell matrix to undergo a transition from a lamellar to a nonlamellar state. Although equilibrium properties of membranes are amenable to detailed theoretical studies, collective rearrangements involved in phase transitions have thus far only been modeled on a qualitative level. Here, for the first time, the complete transition pathway from a multilamellar to an inverted hexagonal phase is elucidated at near-atomic detail using a recently developed coarse-grained molecular dynamics simulation model. Insight is provided into experimentally inaccessible data such as the molecular structure of the intermediates and the kinetics involved. Starting from multilamellar configurations, the spontaneous formation of stalks between the bilayers is observed on a nanosecond timescale at elevated temperatures or reduced hydration levels. The stalks subsequently elongate in a cooperative manner leading to the formation of an inverted hexagonal phase. The rate of stalk elongation is approximately 0.1 nm ns(-1). Within a narrow hydration/temperature/composition range the stalks appear stable and rearrange into the rhombohedral phase.