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.     

A 2H-NMR study of Acholeplasma laidlawii membranes highly enriched in myristic acid

Myristic acid specifically deuterated at several positions along the acyl chain was biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B to the level of ?90%. ²H-NMR was used to study the molecular order and lipid phase composition of the membranes as a function of temperature. Isolated membranes and intact cells give rise to similar ²H spectra. Below 25°C the spectra exhibit a broad gel phase component which at 0°C reaches the rigid limit value expected for an immobilized methylene group. Spectral moments were used to determine the relative amounts of gel and liquid crystalline phase lipids throughout the gel-liquid crystal phase transition. The results indicate that at the growth temperature (37 or 30°C) the A. laidlawii B membrane lipids are ～85–90% in the gel state, and that protein has little effect on lipid order of the liquid crystalline lipid, but leads to an increase in the linewidth by approx. 20%.     

Effects of cholesterol on the orientational order of unsaturated lipids in the membranes of Acholeplasma laidlawii: A 2H-NMR study

We have investigated by ²H-NMR the effects of the incorporation of cholesterol on the orientational order of unsaturated lipid acyl chains in the membranes of Acholeplasma laidlawii B. This is the only ²-NMR study to date of the influence of cholesterol in a biological membrane using specifically labelled fatty acids. We observed the characteristics condensing effect of cholesterol on the lipid acyl chain order in the liquid crystalline phase. In terms of the percentage increase in the quadrupolar splittings, the presence of cholesterol has its greatest effect on the methyl end of the labelled oleoyl chains, with a maximum at the C-14 segment. In absolute terms, the perturbation is greatest in the carboxyl end of the chains. The temperature dependence of the ²H spectra for the cholesterol-containing membranes is very similar to that for the cholesterol-free membranes. The broad phase transition of the membrane lipids, which is characteristic for the samples lacking cholesterol, is apparently little affected by the presence of up to 27 mol% cholesterol. In addition, the temperature of onset of the phase transition is not significantly depressed by the presence of cholesterol.     

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.     

Water binding and phase structures for different Acholeplasma laidlawii membrane lipids studied by deuteron nuclear magnetic resonance and x-ray diffraction

Water binding capability and phase structures for different lipid species extracted from Acholeplasma laidlawii A membranes have been studied using deuteron nuclear magnetic resonance and low-angle X-ray diffraction.The dominating membrane lipids are monoglucosyldiglyceride and diglucosyldiglyceride and each of them takes up limited amounts of water (bound plus trapped), i.e., up to 13% (w/w), whereas the phospholipids and phosphoglycolipids have larger hydration capacities.Addition of magnesium and calcium ions, but not sodium ions, to the diglucosyldiglyceride increases the hydration capability. This increase is accompanied by the formation of a metastable liquid crystalline phase and a hysteresis effect for the transition temperature.Large differences in water deuteron quadrupole splitting were observed between mono- and diglucosyldiglyceride. Both ²H nuclear magnetic resonance and low-angle X-ray diffraction studies on lipids containing biosynthetically incorporated ω-d₃-palmitic acid clearly indicate the existence of a reverse hexagonal phase structure for the monoglucosyldiglyceride and lamellar structures for the diglucosyldiglyceride and the other membrane lipids.The low hydration capability of the large diglucosyldiglyceride polar head is discussed in terms of polar head configuration.Both mono- and diglucosyldiglyceride have several physical properties similar to those of phosphatidylethanolamine.     

The effects of hydration and divalent cations on lamellar-nonlamellar phase transitions in membranes and total lipid extracts from Acholeplasma laidlawii A-EF22 – a 2H NMR study

Acholeplasma laidlawii strain A-EF22 was grown in a medium supplemented with 75 μm α-deuterated palmitic acid (16:0-d ₂) and 75 μm α-deuterated oleic acid (18:1c-d ₂), or with 150 μm 18:1c-d ₂. The fatty acids were incorporated into the membrane lipids and ²H NMR spectra were recorded from intact membranes, total lipid extracts, and the combined glucolipid and neutral lipid fractions of a total lipid extract. The lipids in intact membranes form a bilayer structure up to at least 70 °C. The same result was obtained with membranes digested with pronase, which removes a large fraction of the membrane proteins. A reversed hexagonal liquid crystalline (H_II) phase was formed below 70 °C by the total lipid extracts hydrated with 20 and 30% (w/w) water; in the presence of 40% (w/w) water only one of the extracts formed an H_II phase below 70 °C. The H_II phase was formed at higher temperatures with an increasing water content. However, only a lamellar liquid crystalline (L _α ) phase was formed up to 70 °C by the total lipid extracts when the water concentrations were 50% (w/w) or higher. The temperature (T _LH) for the L _α to H_II phase transition in the combined glucolipid and neutral lipid fractions was only 2–3 °C lower than for the total lipids, and the phospholipids thus have a very modest influence on the T _LH value. Physiologically relevant concentrations of Ca²⁺ and Mg²⁺ ions did not affect the phase equilibria of total lipid extracts significantly. It is concluded from comparison with published data that the membrane lipids of the cell wall-less bacterium A. laidlawii have a smaller tendency to form reversed nonlamellar phases than the membrane lipids of three bacterial species surrounded by a cell wall. Received: 10 March 1997 / Accepted: 4 July 1997     

Studies in membrane processes X: A deuterium magnetic resonance study of dipalmitoyl lecithin and palmitic acid guests in magnetically-oriented hexadecyltrimethyl-ammonium bromide liquid crystalline system

The membrane phospholipid, dipalmitoyl lecithin, deuterium labelled in its fatty acyl chains, and palmitic acid-d₃₁ have been incorporated into the same bilayer model membrane, a lyotropic liquid crystalline hexadecyltrimethylammonium bromide mesophase which spontaneously orients in an applied magnetic field. The order parameter profiles for the lecithin and palmitic acid, and that of the host detergent are quite different indicating that the ordering of the incorporated lipids is not dictated by surrounding detergent molecules, but rather the order imposed is a function of the nature of chemical anchoring of the hydrophilic headgroups of the individual molecules at the bilayer interface. Dissimilarities in the order profiles are interpreted in terms of variations in the case of formation of random gauche conformers along the length of the acyl chains. In addition, the validity of the use of perdeuterated fatty acids as probes of the order of other membrane components is questioned. For the host detergent the relaxation rate ‘$\text{1}\text{T′}{}_2$ obtained from the line width is directly proportional to the deuterium quadrupole splitting of the —CD₂ — segments for that flexible part of the chain beyond the plateau region of constant degree of order. This indicates that for an aligned chain, the increasing motional freedom for each successive segment toward the chain end, which is linked with the increasing probability of single gauche rotations, is the motion responsible for both the decrease in degree of order and thus the increase in line width.     

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°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°C, but in a liquid crystalline state when cells were grown at 37 and 43°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.     

Physical studies of cell surface and cell membrane structure. Deuterium nuclear magnetic resonance investigation of deuterium-labelled N-hexadeconoylgalactosylceramides (cerebrosides)

1. Deuterium Fourier transform nuclear magnetic resonance spectra of a series of N-palmitoylgalactosylceramides (cerebrosides) specifically labelled with deuterium at one of positions 2', 6', 10' and 16' of the acyl chain, or in the C-6 hydroxymethyl group of the galactose residue, have been obtained using a spin-echo technique at 34.1 MHz with a homebuilt superconducting magnet spectrometer. 2. The effects of temperature and cholesterol on the deuterium spectra have been investigated. The results indicate, when compared at the same reduced temperature, that the hydrocarbon chain organization in the liquid crystalline phase of palmitoylgalactosylceramide is essentially identical to that seen in similar chain length glycerophospholipids. In particular, two sets of quadrupole splittings are seen for a 2'-labelled N-palmitoylgalactosylceramide, indicating non-equivalent deuterons as noted previously for phospholipids. 3. Two sets of quadrupole splittings are observed for the headgroup C-6-labelled N-palmitoylgalactosylceramide. It is proposed that these signals arise from the enantiomeric R and S lipids, and that motion of the hydroxymethyl group is slow (greater than 10(-5) S). These results suggest the presence of a hydrogen bond network in the polar headgroup region. 4. The effects of cholesterol on the deuterium spectra of N-palmitoylgalactosylceramide-labelled as C2H3 in the terminal methyl group, at 1:1 mol ratios and in excess water below the crystal to liquid-crystal phase transition temperature (Tc) of the pure lipid (82 degrees C), are different to the effects seen with the phosphatidylcholine-cholesterol system. The spectra below Tc are characterised by two overlapping powder patterns, one with a quadrupole splitting of approx. 6 kHz (fluid liquid-crystalline phase) and one with a quadrupole splitting of about 20--25 kHz (crystal or gel-state lipid). Exchange between these two environments is therefore slow, leading to the possibility of characterising the cerebroside-cholesterol phase diagram using deuterium nuclear magnetic resonance spectroscopy.     

On the interaction between intrinsic proteins and phosphatidylglycerol in the membrane of Acholeplasma laidlawii.

About 30% of the phosphatidylglycerol in oleic acid-enriched Acholeplasma laidlawii membranes are not hydrolyzed at temperatures below 10 °C by phospholipase A₂ from porcine pancreas. Removal of 53% of the membrane proteins by proteolysis did not reduce the size of this inaccessible phosphatidylglycerol pool. However, modification of the membrane proteins with 2,4,6-trinitrobenzenesulfonic acid or glutaraldehyde did make an additional 70% of this protected pool of phosphatidylglycerol accessible to phospholipase A₂. Complete hydrolysis of phosphatidylglycerol at low incubation temperatures was achieved only after heat treatment of the membranes which resulted in an extensive aggregation of intrinsic membrane proteins as visualized by freeze-etch electron microscopy. Phospholipase A₂ from bee venom was more effective in hydrolyzing phosphatidylglycerol at low temperature than the pancreatic enzyme. These results show that the inaccessibility of phosphatidylglycerol is not due to resealing of isolated membranes, the presence of a crystalline phase in the membrane lipids, or a shielding effect of surface proteins. The protection against hydrolysis may be due to an interaction of phosphatidylglycerol with intrinsic membrane proteins which is stabilized at low temperatures. Increasing the temperature favors the exchange of protein-bound phosphatidylglycerol with other membrane lipids resulting in complete hydrolysis.     

The effect of headgroup class on the conformation of membrane lipids in Acholeplasma Laidlawii: A 2H-NMR study

[2-²H₂]Oleic, [2-²H₂]palmitic, [2-²H₂]dihydrosterculic and [3-²H₂]oleic acids were biosynthetically incorporated into the membrane lipids of Acholeplasma laidlawii B. ²H-NMR spectroscopy and spectral ‘de-Parking” (M. Bloom, J.H. Davis and M.I. Valic, Can. J. Phys., 58 (1980) 1510) were used to study the effect of lipid headgroup class on the conformational order in the vicinity of the C-2 position of the acyl chains of lipids in the liquid crystalline phase. The results indicate that although the orientation and conformations of the membrane lipids in the region of the C-2 position of the chains are qualitatively very similar among the various lipid classes, quantitatively there are some differences, particularly between the glycolipids and the phospholipids. These differences do not exted to the C-3 position. Unlike the headgroup class, the membrane proteins appear to have little if any effect on the molecular ordering of the lipids.     

Carbon-13 and proton nuclear magnetic resonance of unsonicated model and mitochondrial membranes

Proton nuclear magnetic resonance (PMR) spectra at 270 MHz of aqueous dispersions of nonsonicated egg lecithin, dipalmitoyl lecithin, egg lecithin-cholesterol (1 : 1) and dipalmitoyl lecithin-cholesterol (1 : 1), together with PMR spectra of mitochondrial membranes and their extracted lipids, have been obtained.Carbon-13 nuclear magnetic resonance (CMR) spectra at 25.2 MHz of egg lecithin, egg lecithin-cholesterol (1 : 1) and sphingomyelin, together with CMR spectra of chloroplast and mitochondrial membranes, and erythrocyte ghosts, have also been obtained. The results obtained using CMR appear very promising for further study of intact membrane structure.It is suggested, on the basis of CMR evidence, that the proteins in mitochondrial membranes may be considerably less mobile than the lipids.     

Temperature-induced vertical shift of proteins in membranes

Thermotropic changes in the transverse order of microsomal membranes isolated from Tetrahymena are revealed by low-angle X-ray diffraction. These are correlated with the lateral order of the membrane lipids by wide-angle X-ray diffraction. Upon lowering the temperature from 28 °C to 2 °C, the Bragg period of the membrane stack reveals an abrupt increase of ~3.0 nm at ~19 °C, which is reversible upon reheating to 28 °C. This is coupled with an alteration in the electron density profile, revealing a shift of mass from the hydrophobic core towards one of the two hydrophilic surfaces. Between 35 °C and 0 °C, the membrane lipids undergo a broad, thermotropic “two-stage” liquid crystalline ? crystalline phase separation with a “breakpoint” at ~18 °C. This breakpoint signals an abrupt lipid redistribution, presumably due to a change in the composition of the two coexisting liquid crystalline and crystalline lipid phases. We conclude: (1) the temperature-induced mass shift reflects a shift in the transverse partition of proteins in membranes; (2) this is triggered by an abrupt lipid redistribution occurring during a broad liquid crystalline ? crystalline phase separation.     

Raman spectroscopy of the milk globule membrane and triglycerides

The acyl chain mobilities of the lipids of bovine milk fat globules and the component triglycerides have been determined by Raman spectroscopy as a function of temperature from -100°C to 80°C. A broad transition is observed centered about 2–6°C. The C-H and C-C stretching bands in the spectra of liquid and crystalline triglycerides are used comparatively to show that the lipids of the milk globule membrane are 30–40% more ordered than the lipids of the intact milk fat globules at 20°C. Synthetic triglyceride melts, quenched rapidly, are used to illustrate the effect of the thermal history of a sample on lipid order as determined spectroscopically.Strong infrared amide I and amide II bands at 1646 and 1543 cm^?1, respectively, indicate that the protein conformation of the globule membrane is not characterized by extensive regions of beta-sheet structure. Raman spectra of the globule triglycerides indicate cis unsaturation of $\text{39 ± 5\%}$ by comparison to triolein and trielaidin.     

Acholeplasma laidlawii membranes: an electron spin resonance study of the influence on molecular order of fatty acid composition and cholesterol.

The effects on membrane structure of including various fatty acids and cholesterol in the growth medium of Acholeplasma laidlawii were investigated by the use of spin-labeled fatty acids. Although the order-mobility parameters varied significantly at some temperatures with the nature of the fatty acid incorporated, the value measured at the growth temperature was only slightly affected by changes in the fatty acid composition of the membranes. The data confirm previous assertions that despite a high level of incorporation of fatty acids of various chain lengths or degree of unsaturation, A. laidlawii regulates its overall membrane fluidity within close limits at the growth temperature. Incorporation of cholesterol increased the degree of order at all temperatures. The coexistence of two lipid phases, one protein-dependent, could be observed in membranes. The order-mobility parameter of spin probes proved less satisfactory for the observation of a gel to liquid crystal transition of the membrane lipid than the partition parameter of a fatty acid spin probe. Order parameters measured by fatty acid spin probes were somewhat higher than those measured by the analogous ²H nmr probes.     

Deuterium Magnetic Resonance Studies of Senescence-Related Changes in the Physical Properties of Rose Petal Membrane Lipids

The physical properties of membrane lipids in senescing rose (Rosa hybrida L., cv Mercedes) petals were studied by deuterium nuclear magnetic resonance (2H-NMR) and fluorescence depolarization. All of the 2H-NMR spectra arising from deuterated dimyristoylphosphatidylcholine mixed with whole-lipid extracts from membranes of petals of different ages had a shape that is characteristic of liquid-crystalline lipid at 30[deg]C. Arrhenius plots of the moments of the 2H spectra and fluorescence depolarization values measured from 1,6-diphenyl hexatriene-labeled rose petal membrane lipid samples indicated that membrane lipid order increased with decreasing temperature as well as with increasing age of the petals. The latter trend is explained by previously observed increases in fatty acid saturation and increases in the sterol-to-phospholipid ratio that occur in rose petals during senescence. The 2H-NMR spectra obtained at 0[deg]C also contained quadrupolar splitting lines from lipid in the gel phase, confirming the occurrence of this phase in membranes from this tissue.