The temperature dependence of molecular order and the influence of cholesterol in Acholeplasma laidlawii membranes

²H nuclear magnetic resonance (NMR) of Acholesplasma laidlawii membranes grown on a medium supplemented with perdeuterated palmitic acid shows that at 42°C or above, the membrane lipids are entirely in a fluid state, exhibiting the characteristic ‘plateau’ in the variation of deuterium quadrupolar splitting with chain position. Between 42 and 34°C there is a well-defined gel-to-fluid phase transition encompassing the growth temperature of 37°C, and at lower temperatures the membranes are in a highly ordered gel state. The ²H-NMR spectra of the gel phase membranes are similar to those of multilamellar dispersions of chain perdeuterated dipalmitoyl phosphatidylcholine (Davis, J.H. (1979) Biophys. J. 27, 339) as are the temperature dependences of the spectra and their moments. The incorporation of large amounts of cholesterol into the membrane removes the gel to fluid phase transition. Between 20 and 42°C, the position dependence of the orientational order of the hydrocarbon chains of the membranes is similar to that of the fluid phase of the membranes without cholesterol, i.e., they exhibit the plateau in the deuterium quadrupolar splittings. However, the cholesterol-containing membranes have a higher average order, with the increases in order being greater for positions near the carbonyl group of the acyl chains. Below 20°C the ²H spectra of the membranes containing cholesterol change dramatically in a fashion suggestive of complex motional and/or phase behaviour.     

A deuterium NMR study of labelled n-alkanol anesthetics in a model membrane

The 2H-NMR spectra of 50 wt.% aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) containing either selectively deuterated 1-decanol (25 mol%) or [2H17]-1-octanol (25 mol%) have been measured as a function of temperature. Both alkanols are potent anesthetics. A detailed carbon-deuterium bond order parameter profile of 1-decanol in liquid crystalline phospholipid dispersions at 50 degrees C was determined from the quadrupolar splittings of 1-decanols deuterated at eight different positions. A maximum order parameter SCD = 0.20 was obtained for [5,5-2H2]-1-decanol, with labels at both ends of the 1-decanol exhibiting reduced order parameters. Explanations for the reduced order towards the hydroxyl group of 1-decanol are discussed in terms of either increased amplitudes of motion or geometric effects due to hydrogen bonding. By comparing the order parameter profile of sn-2 chain deuterated phosphatidylcholine dispersions containing 25 mol% 1-decanol (J.L. Thewalt, S.R. Wassall, H. Gorrissen and R.J. Cushley, Biochim. Biophys. Acta, 817 (1985) 355) with the profile of deuterated 1-decanol in DPPC, we estimate that decanol is approximately parallel to the C-3 to C-13 region of the phosphatidylcholine's sn-2 chain. Variation of the spectral moments M1 with temperature indicates that both 1-decanol and 1-octanol are sensitive to the packing of the lipid in which they are dissolved. Below the phase transition temperature, the 2H-NMR spectra of either 1-decanol (selectively deuterated) or 1-octanol (perdeuterated) are broad powder patterns, characteristic of axially symmetric rotation about the alcohol's long axis. This is in contrast to the 2H-NMR spectra obtained from deuterated phosphatidylcholine under similar conditions, which implies that the phospholipid acyl chain conformations are more restricted than those of the alcohol at these temperatures. From the M1 behavior of the various alkanol chain segments with temperature, the gel to liquid crystalline phase transition is seen to initiate in the middle of the DPPC/1-alkanol bilayer.     

Study of the cytoplasmic and outer membranes of Escherichia coli by deuterium magnetic resonance.

The cytoplasmic and outer membranes of Escherichia coli were studied between 0 and 40 degrees C by deuterium magnetic resonance quadrupolar echo spectroscopy. The L51 strain of E. coli was used to incorporate perdeuterated palmitic acid into the membrane phospholipids. The cytoplasmic and outer membranes were separated using standard techniques. The spectrum of each membrane preparation was dominated at high temperatures (greater than or equal to 37 degrees C) by the characteristic liquid-crystalline plateau previously observed for perdeuterated palmitate chains in model phospholipid membranes. At low temperatures, the shape and width of the spectrum were characteristic of the gel phase. The relative intensities of the liquid-crystalline and gel features varied systematically with temperature. A quantitative analysis of the acyl chain orientational order was carried out by using the method of moments. The orientational order at each temperature was greater in the outer membrane sample than in that of the cytoplasmic membrane, indicating that the liquid-crystalline-gel transition region in the outer membrane is shifted to higher temperatures than that of the cytoplasmic membrane by about 7 degrees C. It is clear from the results that most of the phospholipid molecules participate in the phase transition.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems.

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions. The lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (-120 degrees C to +120 degrees C). Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids. Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H2O and 2H2O media yielded a value of 1.013 +/- 0.026 ml/g for the partial specific volume of this lipid. We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude. Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

Deuterium NMR study of the effect of n-alkanol anesthetics on a model membrane system

The effects of 25 mol% incorporation of two anesthetics, 1-octanol and 1-decanol, on a deuterated, saturated phospholipid in 50 wt% aqueous multilamellar dispersions have been studied by 2H-NMR spectroscopy and differential scanning calorimetry (DSC). The phospholipid used is sn-2 substituted '[2H31]-palmitoylphosphatidylcholine' (PC-d31). DSC thermograms demonstrate that PC-d31 has phase behavior qualitatively similar to that of dipalmitoylphosphatidylcholine, with a pretransition at 31 degrees C and a main gel to liquid crystalline transition at 40 degrees C. Analysis of the temperature-dependent 2H-NMR spectra in terms of the first moment, which is extremely sensitive to the phospholipid phase, shows that 1-octanol and 1-decanol depress and broaden the main transition. This is confirmed by DSC, which shows that the pretransition is eliminated by the 1-alkanols. The carbon-deuterium bond order of the phospholipid deuterated acyl chains, in the presence and absence of 1-alkanols, was determined from deuterium quadrupolar splittings. Spectra were analyzed using the depaking technique. A 1-alkanol concentration of 25 mol% had no significant effect on the profile of the carbon-deuterium bond order parameter SCD along the phospholipid acyl chain at 50 degrees C. Thus, it appears that the liquid crystalline phase is able to accommodate large amounts of linear anesthetic molecules without substantial effect on molecular ordering within the membrane bilayer. Preliminary results show that the transverse relaxation rates of the acyl chain segments are significantly decreased by the presence of 1-octanol or 1-decanol.     

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.     

Deuterium NMR investigation of ether- and ester-linked phosphatidylcholine bilayers

Deuterium nuclear magnetic resonance (2H NMR) spectra of specifically head-group- and chain-deuterated ester- and ether-linked phosphatidylcholine bilayers were studied as a function of temperature over the range -33 to 50 degrees C. Head-group-deuterated dihexadecylphosphatidylcholine ([alpha-2H2]DHPC) bilayers yield line shapes and spin-lattice relaxation times similar to those observed for its ester-linked counterpart, dipalmitoylphosphatidylcholine ([alpha-2H2]DPPC), in the high-temperature ripple and L alpha bilayer phases. These results indicate the ether linkage has no effect on the dynamics or the orientational order at the alpha-C2H2 segment of the phosphocholine head group. At all temperatures, the 2H NMR spectra of chain-deuterated 1,2[1',1'-2H2]DHPC bilayers exhibit a reduced spectral width compared to 1,2[2',2'-2H2]DPPC bilayers. The most significant feature of the deuterated alkyl chain spectrum of DHPC at 45 degrees C is the observation of four separate quadrupolar splittings from the alpha-methylene segments of the alkyl chains, in comparison to the three quadrupolar splittings reported previously from the alpha-methylene segments of the acyl chains of DPPC. Spin-lattice relaxation experiments performed on DHPC suggest an assignment of the two smaller and the two larger quadrupolar splittings to separate alkyl chains, respectively. Low-temperature (T less than or equal to -20 degrees C) gel-phase spectra of deuterated head-group [alpha-2H2]DHPC remain an order of magnitude narrower than those observed for [alpha-2H2]DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

High pressure 2H nuclear magnetic resonance study of the gel phases of dipalmitoylphosphatidylcholine

The 2H-NMR lineshapes of dipalmitoylphosphatidylcholine perdeuterated in the acyl chains were studied in a 15% dispersion in water as a function of pressure from 1 bar to 5 kbar over the temperature range from 7 degrees C to 75 degrees C. Increasing pressure in the gel state had the same effect as lowering the temperature: the lineshape gradually changed from a motionally averaged to a rigid lattice type spectrum with much of the intensity in the shoulders at +/- 63 kHz. At very high pressures and low temperatures (7 degrees C, 2.5 kbar; 25 degrees C, 5 kbar) even the methyl portion of the spectrum became a rigid lattice type spectrum at +/- 21 kHz. In addition to the liquid crystalline phase, five gel phases were detected. Using different techniques to determine the phase transitions, a general pressure-temperature phase diagram was constructed.     

Physicochemical characterization of 1,2-diphytanoyl-sn-glycero-3-phosphocholine in model membrane systems

We report here on a series of studies aimed at characterization of the structural and dynamical properties of the synthetic lipid diphytanoyl phosphatidylcholine, in multilamellar dispersions and vesicle suspensions.This lipid exhibits no detectable gel to liquid crystalline phase transition over a large temperature range (?120°C to +120°C).Examination of proton nuclear magnetic resonance (NMR) free induction decays obtained from multilayer dispersions of diphytanoyl phosphatidylcholine provided an estimate of the methylene proton order parameter. The estimated magnitude of 0.21 is comparable to those determined for other phospholipids.Sonication of aqueous dispersions of diphytanoyl phosphatidylcholine led to formation of bilayer vesicles as determined by the measurement of the outer/inner choline methyl proton resonances, vesicle sizes in electron micrographs, and comparison of proton NMR linewidths between multilayer and sonicated dispersions. Ultracentrifugation studies of diphytanoyl phosphatidylcholine vesicles in H²O and ²H₂O media yielded a value of 1.013 ± 0.026 ml/g for the partial specific volume of this lipid.We have measured spin lattice relaxation rates for the methyl and methylenemethyne protons of the hydrocarbon chains of diphytanoyl phosphatidylcholine in bilayer vesicles over a range of temperatures and at two NMR frequencies (100 and 220 MHz). The observed relaxation rates for the methylene protons in this system were approximately twice those previously reported for dipalmitoyl phosphatidylcholine at comparable temperatures and resonance frequencies, whereas the relaxation rates measured for the methyl protons were greater than those of the straight chain lipid by an order of magnitude.Measurement of the spin lattice relaxation rates of the hydrocarbon protons of the diphytanoyl phosphatidylcholine in a 10 mol% mixture of the branched-chain lipid in a deuterated host lipid, diperdeuteropalmitoyl phosphatidylcholine, showed a discontinuity in the temperature dependence of the proton NMR longitudinal relaxation rates of the branched-chain lipid in the region of the gel to liquid crystalline phase transition temperature of the deuterated dipalmitoyl phosphatidylcholine host lipid. This result may be taken as evidence of lateral phase separation of a liquid cyrstalline phase enriched in diphytanoyl phosphatidylcholine from a gel phase enriched in diperdeuteropalmitoyl phosphatidylcholine at temperatures below the phase transition temperature of deuterated host lipid. This conclusion is supported by the observation of an abrupt change in the hydrocarbon methylene linewidth (at 100 MHz) of 10 mol% diphytanoyl phosphatidylcholine in diperdeuteropalmitoyl phosphatidylcholine over the temperature range where lateral phase separation is taking place according to differential thermograms.     

A Fourier transform infrared spectroscopic study of the interaction of alkaline earth cations with the negatively charged phospholipid 1, 2-dimyristoyl-sn-glycero-3-phosphoglycerol

The interaction of aqueous phospholipid dispersions of negatively charged 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol, sodium salt (DMPG) with the divalent cations Mg(2+), Ca(2+) and Sr(2+) at equimolar ratios in 100 mM NaCl at pH 7 was investigated by Fourier transform infrared spectroscopy. The binding of the three cations induces a crystalline-like gel phase with highly ordered and rigid all-trans acyl chains. These features are observed after storage below room temperature for 24 h. When the gel phase is heated after prolonged incubation at low temperature phase transitions into the liquid crystalline phase are observed at 58 degrees C for the DMPG:Sr(2+), 65 degrees C for the DMPG:Mg(2+), and 80 degrees C for the DMPG:Ca(2+) complex. By subsequent cooling from temperatures above T(m) these complexes retain the features of a liquid crystalline phase with disordered acyl chains until a metastable gel phase is formed at temperatures between 38 and 32 degrees C. This phase is characterized by predominantly all-trans acyl chains, arranged in a loosely packed hexagonal or distorted hexagonal subcell lattice. Reheating the DMPG:Sr(2+) samples after a storage time of 2 h at 4 degrees C results in the transition of the metastable gel to the liquid crystalline phase at 35 degrees C. This phase transition into the liquid crystalline state at 35 degrees C is also observed for the Mg(2+) complex. However, for DMPG:Mg(2+) at higher temperatures, a partial recrystallization of the acyl chains occurs and the high temperature phase transition at 65 degrees C is also detected. In contrast, DMPG:Ca(2+) exhibits only the phase transition at 80 degrees C from the crystalline gel into the fluid state upon reheating. Below 20 degrees C, the rate of conversion from the metastable gel to a thermodynamically stable, crystalline-like gel phase decreases in the order Ca(2+)&z. Gt;Mg(2+)>Sr(2+). This conversion into the crystalline gel phase is accompanied by a complete dehydration of the phosphate groups in DMPG:Mg(2+) and by a reorientation of the polar lipid head groups in DMPG:Ca(2+) and in DMPG:Sr(2+). The primary binding sites of the cations are the PO(2)(-) groups of the phosphodiester moiety. Our infrared spectroscopic results suggest a deep penetration of the divalent cations into the polar head group region of DMPG bilayers, whereby the ester carbonyl groups, located in the interfacial region of the bilayers, are indirectly affected by strong hydrogen bonding of immobilized water molecules. In the liquid crystalline phase, the interaction of all three cations with DMPG is weak, but still observable in the infrared spectra of the DMPG:Ca(2+) complex by a slight ordering effect induced in the acyl chains, when compared to pure DMPG liposomes.     

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.     

A calorimetry and deuterium NMR study of mixed model membranes of 1-palmitoyl-2-oleylphosphatidylcholine and saturated phosphatidylcholines

Binary phase diagrams have been constructed from differential scanning calorimetry (DSC) data for the systems 1-palmitoyl-2-oleylphosphatidylcholine (POPC)/dimyristoylphosphatidylcholine (DMPC), POPC/dipalmitoylphosphatidylcholine (DPPC) and POPC/distearoylphosphatidylcholine (DSPC). Mixtures of POPC with DMPC exhibit complete miscibility in the gel and liquid crystalline states. Mixtures of POPC with DPPC or with DSPC exhibit gel phase immiscibility over the composition range 0-75% DPPC (or DSPC). These results, when taken together with previous studies of mixtures of phosphatidylcholines, are consistent with the hypothesis that PCs whose order-disorder transition temperatures (Tm values) differ by less than 33 deg. C exhibit gel state miscibility. Those whose Tm values differ by more than 33 deg. C exhibit gel state immiscibility. 2H-NMR spectroscopy has been used to further study mixed model membranes composed of POPC and DPPC, in which either lipid has been labeled with deuterium in the 2-, 10- or 16-position of the palmitoyl chain(s) or in the N-methyls of the choline head group. POPC/DPPC mixtures in the liquid crystalline state are intermediate in order between pure POPC and DPPC at the same temperature. The POPC palmitoyl chain is always more disordered than the palmitoyl chains of DPPC in liquid crystalline POPC/DPPC mixtures. This is attributed to the fact that a POPC palmitoyl chain is constrained by direct bonding to have at least one oleyl chain among its nearest neighbors, while a DPPC palmitoyl chain must have at least one neighboring palmitoyl chain. When liquid crystalline POPC, DPPC and POPC/DPPC mixtures are compared at a reduced temperature (relative to the acyl chain order-disorder transition), POPC/DPPC mixtures are more disordered than predicted from the behavior of the pure components, in agreement with enthalpy data derived from DSC studies. Within the temperature range of the broad phase transition of 1:1 POPC/DPPC, a superposition of gel and liquid crystalline spectra is observed for 1:1 POPC/[2H]DPPC, while 1:1[2H]POPC/DPPC exhibits only a liquid crystalline spectrum. Thus, at temperatures within the phase transition region, the liquid crystalline phase is POPC-rich and the gel phase is DPPC-rich. Comparison of the liquid crystalline quadrupole splittings within the thermal phase transition range suggests that mixing of the residual liquid crystalline POPC and DPPC is highly non-ideal.     

Analysis of the defect structure of gel-phase lipid.

The partitioning of the spin label 2,2,6,6-tetramethylpiperidinyl-1-oxy (Tempo) into phosphatidylcholine bilayers and the monomer-aggregate equilibrium for chlorophyll a incorporated into phosphatidylcholine bilayers have been interpreted in terms of the formation of defects in the gel-phase lipid, starting some 20 degrees C below the temperature of the main gel to liquid crystalline phase transition. By contrast, defects seem to be largely absent from bilayers of dipalmitoylphosphatidylethanolamine in the gel phase. The defect structure accounts for the continuous nature of the phase transition for phosphatidylcholines, and also for the increase in width of the transition caused by the addition of alcohols.     

A new crystalline phase of L-alpha-dipalmitoyl phosphatidylcholine monohydrate.

A new phase transition of L-alpha-dipalmitoyl phosphatidylcholine (DPPC) monohydrate from the "biaxial" phase to a crystalline phase (C phase) has been found at 71 degrees C by means of infrared attenuated total reflection (IR-ATR) spectroscopy. The transition is characterized by drastic conformational changes in the glycerophosphorylcholine moiety, which led on the one hand to an alignment of the turn near the ester group in the hydrocarbon chain at glycerol C(2) position. On the other hand a uniform conformation of the glycerophosphorylcholine moiety is found to be typical for the C phase, in contrast to nonuniform head group conformations of DPPC in other regions of the DPPC/water phase diagram investigated so far.     

Action of cobra venom phospholipase A2 on the gel and liquid crystalline states of dimyristoyl and dipalmitoyl phosphatidylcholine vesicles

The activity of phospholipase A2 from cobra venom toward phospholipid in single-walled, sonicated vesicles was analyzed, particularly with respect to its activity toward the saturated phosphatidylcholines in the gel and liquid crystalline states. When egg phosphatidylcholine vesicles are used as substrate, the phospholipase has an apparent Km of 4.4 mM, an apparent Vmax of 100 mumol min-1 mg-1 of protein, and a pH optimum of 5.0 at 40 degrees C. The phospholipase hydrolyzed the gel state of dimyristoyl phosphatidylcholine vesicles and dipalmitoyl phosphatidylcholine vesicles at a rate 2 to 3 times greater than the liquid crystalline state, taking into account temperature effects on the enzymatic reaction itself. The results suggest that, toward sonicated vesicles, there is no specific enhancement of the rate when the both liquid crystalline and gel states are present together, as has been suggested to occur for multibilayers studied with other phospholipases. An apparent stimulation of activity as the reaction proceeded was observed above the phase transition temperature. This might be attributed to an increase in the phase transition temperature caused by free fatty acids so that, in the presence of reaction products, the enzyme is actually hydrolyzing gel state phospholipid which was found to be the preferred lipid state for phospholipase activity.     

Relationship of growth temperature and thermotropic lipid phase changes in cytoplasmic and outer membranes from Escherichia coli K12.

Purified cytoplasmic and outer membranes isolated from cells of wild type Escherichia coli grown at 12, 20, 37 and 43 degrees C were labelled with the fatty acid spin probe 5-doxyl stearate. Electron spin resonance spectroscopy revealed broad thermotropic phase changes. The inherent viscosity of both membranes was found to increase as a function of elevated growth temperature. The lipid order to disorder transition in the outer membrane but not the cytoplasmic membrane was dramatically affected by the temperature of growth. As a result, the cytoplasmic membrane presumably existed in a gel + liquid crystalline state during cellular growth at 12 and 20 degrees C, but in a liquid crystalline state when cells were grown at 37 and 43 degrees C. In contrast, the outer membrane apparently existed in a gel + liquid crystalline state at all incubation temperatures. Data presented here indicate that the temperature range over which the cell can maintain the outer membrane phospholipids in a mixed (presumedly gel + liquid crystalline) state correlates with the temperature range over which growth occurs.     

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.     

Factors affecting the motion of the polar headgroup in phospholipid bilayers. A 31P NMR study of unsonicated phosphatidylcholine liposomes.

(1) The 129 MHZ and 36.4 MHZ 31 P NMR spectra of unsonicated liposomes consisting of phosphatidylcholines of varying chain length and unsaturation have been investigated. (2) In the liquid crystalline state the 31 P NMR liposome spectra are similar for both saturated and unsaturated phosphatidylcholines, demonstrating that the motion of the polar headgroup is not sensitive to the fatty acid composition in the disordered liquid crystalline state. (3) Below the hydrocarbon phase transition temperature there is a marked increase in the linewidth of the 31P NMR liposome spectra, indicating a reduction in the motion of the polar headgroup. (4) The addition of equimolar concentrations of cholesterol to phosphatidylcholine eliminates phase transition effects experienced by the polar headgroup. The motion of the polar headgroup is then very similar to that obtained in the liquid crystalline state for pure phosphatidylcholine bilayers. (5) In the liquid crystalline state the motion of the polar headgroup in the phosphate region is insensitive to changes in the available area per phosphatidy-choline molecule.     

A study of mobility and order in model membranes using 2H NMR relaxation rates and quadrupole splittings of specifically deuterated lipids

²H NMR spectra have been observed for several selectively deuterated phospholipid and fatty acid probes intercalated in the liquid crystalline phase of egg phosphatidylcholine in aqueous dispersion. For unsonicated lamellar dispersions and planar multibilayers, quadrupole splittings may be observed which lead directly to a value for the order parameter for the carbon-deuterium bond. Sonicated dispersions yield high-resolution spectra, from which spin-lattice relaxation rates and correlation times for rotational diffusion can be obtained. The presence of cholesterol in the dispersion has no effect on the quadrupole splittings and relaxation rates for ²H in the choline methyl groups, in contrast to its profound effect on the spectra for ²H in the hydrocarbon chains.     

Hydration and lateral organization in phospholipid bilayers containing sphingomyelin: a 2H-NMR study

Interfacial properties of lipid bilayers were studied by (2)H nuclear magnetic resonance spectroscopy, with emphasis on a comparison between phosphatidylcholine and sphingomyelin. Spectral resolution and sensitivity was improved by macroscopic membrane alignment. The motionally averaged quadrupolar interaction of interlamellar deuterium oxide was employed to probe the interfacial polarity of the membranes. The D(2)O quadrupolar splittings indicated that the sphingomyelin lipid-water interface is less polar above the phase transition temperature T(m) than below T(m). The opposite behavior was found in phosphatidylcholine bilayers. Macroscopically aligned sphingomyelin bilayers also furnished (2)H-signals from the amide residue and from the hydroxyl group of the sphingosine moiety. The rate of water-hydroxyl deuteron exchange could be measured, whereas the exchange of the amide deuteron was too slow for the inversion-transfer technique employed, suggesting that the amide residue is involved in intermolecular hydrogen bonding. Order parameter profiles in mixtures of sphingomyelin and chain-perdeuterated phosphatidylcholine revealed an ordering effect as a result of the highly saturated chains of the sphingolipids. The temperature dependence of the (2)H quadrupolar splittings was indicative of lateral phase separation in the mixed systems. The results are discussed with regard to interfacial structure and lateral organization in sphingomyelin-containing biomembranes.