Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.     

Selective Acylation of Plasma Membrane Proteins of Mycoplasma mycoides subsp. mycoides SC,the Contagious Bovine Pleuropneumonia Agent

The plasma membrane of Mycoplasma mycoides subsp. mycoides SC (strain KH₃J) contains over 160 polypeptides with apparent molecular masses ranging from 14 to 125 kDa and isoelectric point values (pIs) from 5 to 9. In vivo labeling with [¹⁴C]-fatty acids revealed about 35 acylated polypeptides including the two major components p42 and p65 and displaying pIs between 5.5 and 9.0, with a majority between 6.5 and 8. The amphiphilic nature of most of these acyl proteins was confirmed by Triton X-114 phase partitioning. Gas-liquid chromatography analyses showed that the order of preference for protein acylation was 16:0 > 18:2c > 18:1c > 18:0 > 14:0, with 16:0 being the major O-ester-bound fatty acyl chain and 18:2c the major N-linked chain. The presence of S-glycerylcysteine and a ratio of [O-ester-bound acyl chains + N-linked chains]/O-ester bound chains of ≈ 1.2 in M. mycoides subsp. mycoides SC membrane proteins are consistent with a lipid modification similar to that occurring in lipoproteins of Gram-negative eubacteria that contain an N-terminal acyl S-glycerylcysteine.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Selective Acylation of Plasma Membrane Proteins of Mycoplasma agalactiae: The Causal Agent of Agalactia

Revealed by in vivo labeling with ¹⁴C-palmitic acid, about 15 acylated proteins were identified in the plasma membrane of Mycoplasma agalactiae (type strain PG2), including the major component p40. Triton X-114 phase partitioning and Western blotting demonstrated the amphiphilic properties of the acyl proteins and showed that they were also antigenic components. Chemical analyses of fatty acids bound to proteins revealed the following selectivity order within acylation: stearic acid (18:0) > linoleic acid (18:2c) ≈ palmitic acid (16:0) > oleic acid (18:1c) > myristic acid (14:0), with 16:0 and 18:1c preferred for the O-acylation and 18:0 for the N-acylation. The ratio [O-ester- + amide-bound acyl chains]/O-ester-linked chains being close to 1.4 as well as the presence of S-glycerylcysteine suggest that acyl proteins in M. agalactiae are true lipoproteins containing N-acyl diacyl glycerylcysteine, probably processed by a mechanism analogous to that described for Gram-negative eubacteria. Received: 22 June 1999 / Accepted: 2 August 1999     

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.     

Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii.

Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins. The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains. Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively. No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids. Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates. The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins. Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114. This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.     

The assimilation of glycerol into lipid acyl chains and associated carbon backbones of Nannochloropsis salina varies under nitrogen replete and deplete conditions

Mixotrophic cultivation can increase microalgae productivity, yet the associated lipid metabolism remains mostly unknown. Stable isotope labeling was used to track assimilation of glycerol into the triacylglyceride (TAG) and membrane lipids of Nannochloropsis salina. In N-replete media, glycerol uptake and ¹³C incorporation into acyl chains were, respectively, 6-fold and 12-fold higher than in N-deplete conditions. In N-replete cultures, 42% of the carbon in the consumed glycerol was assimilated into lipid acyl chains, mostly in membrane lipids rather than TAG. In N-deplete cultures, only 11% of the limited amount of consumed glycerol was fixed into lipid acyl chains. Labeled lipid-associated glycerol backbones were predominantly ¹³C₃ labeled, suggesting that intact glycerol molecules were directly esterified with fatty acids/polar head groups. However, the presence of singly and doubly labeled lipid-bound glycerol species suggested that some glycerol also went through the central carbon metabolism before forming glycerol-3-phosphate destined for lipid esterification. ¹³C incorporation was higher in the saturated and monounsaturated than the polyunsaturated acyl chains of TAG, indicating the flux of carbon from glycerol went first to de novo fatty acid synthesis before acyl editing reactions. The results demonstrate that nitrogen availability influences both glycerol consumption and utilization for lipid synthesis in Nannochloropsis, providing novel insights for developing mixotrophic cultivation strategies.     

Biophysical and biochemical mechanisms by which dietary N-3 polyunsaturated fatty acids from fish oil disrupt membrane lipid rafts

N-3 polyunsaturated fatty acids (PUFAs) from fish oil exert their functional effects by targeting multiple mechanisms. One mechanism to emerge in the past decade is the ability of n-3 PUFA acyl chains to perturb the molecular organization of plasma membrane sphingolipid/cholesterol-enriched lipid raft domains. These domains are nanometer-scale assemblies that coalesce to compartmentalize select proteins for optimal function. Here we review recent evidence on how n-3 PUFAs modify lipid rafts from biophysical and biochemical experiments from several different model systems. A central theme emerges from these studies. N-3 PUFA acyl chains display tremendous conformational flexibility and a low affinity for cholesterol and saturated acyl chains. This unique flexibility of n-3 PUFA acyl chains impacts the organization of inner and outer leaflet lipid rafts by disrupting acyl chain packing and molecular order within rafts. Ultimately, the disruption in raft organization has consequences for protein clustering and thereby signaling. Overall, elucidating the complex mechanisms by which n-3 PUFA acyl chains reorganize membrane architecture will enhance the translation of these fatty acids into the clinic for treating several diseases.     

Changes in fatty acids composition,hydrogen peroxide generation and lipid peroxidation of salt-stressed corn (<Emphasis Type="Italic">Zea mays</Emphasis> L.) roots

Comparative study about the salt-induced oxidative stress and lipid composition has been realised in primary root tissues for two varieties of maize (Zea mays L.) in order to evaluate their responses to salt stress. The root growth, root water content (WC), hydrogen peroxide (H₂O₂) generation, lipid peroxidation, membrane stability index and the changes in the profile of fatty acids composition were investigated. Salinity impacts in term of root growth, water content, H₂O₂ generation, lipid peroxidation and membrane destabilisation were more pronounced in primary roots of Aristo than in those of Arper indicating more sensitivity of the first variety. It was confirmed by gas chromatography that the composition of fatty acids in roots of both varieties was constituted mainly by 16:0 and 18:0 as major saturated fatty acids and 18:1ω9, 18:2ω6 and 18:3ω3 as major unsaturated fatty acids. Total lipid extracts from the roots of both varieties showed that the lipid saturation level increased under salt stress, notwithstanding the increased proportion of polyunsaturated fatty acids. The changes in lipid saturation being predominantly due to decreases in oleic acid (18:1ω9) and increases in palmitic acid (16:0). However, Arper root extracts contained a lower proportion of saturated lipids than Aristo. The enhanced proportion of highly polyunsaturated fatty acids especially linolenic and eicosapentaenoic acids was considered to be the characteristic of the relatively salt tolerance in Arper roots.     

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.     

Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations

Understanding how wheat (Triticum aestivum L.) plants under high temperature (HT) regulate lipid composition is critical to developing climate‐resilient varieties. We measured 165 glycerolipids and sterol derivatives under optimum and high day and night temperatures in wheat leaves using electrospray ionization‐tandem mass spectrometry. Levels of polar lipid fatty acyl chain unsaturation were lower in both heat‐tolerant genotype Ventnor and susceptible genotype Karl 92 under HT, compared with optimum temperature. The lower unsaturation was predominantly because of lower levels of 18:3 acyl chains and higher levels of 18:1 and 16:0 acyl chains. Levels of 18:3‐containing triacylglycerols increased threefold/more under HT, consistent with their possible role in sequestering fatty acids during membrane lipid remodelling. Phospholipids containing odd‐numbered or oxidized acyl chains accumulated in leaves under HT. Sterol glycosides (SG) and 16:0‐acylated sterol glycosides (ASG) were higher under HT than optimum temperatures. Ventnor had lower amounts of phospholipids with oxidized acyl chains under HT and higher amounts of SG and 16:0‐ASG than Karl 92. Taken together, the data demonstrate that wheat leaf lipid composition is altered by HT, in which some lipids are particularly responsive to HT, and that two wheat genotypes, chosen for their differing physiological responses to HT, differ in lipid profile under HT.     

Declining viability and lipid degradation during pollen storage

Declining viability of pollen during storage at 24° C in atmospheres of 40% relative humidity (RH) and 75% RH was studied, with special emphasis on lipid changes. Pollens of Papaver rhoeas and Narcissus poeticus, characterized by a high linolenic acid content, were compared with Typha latifolia pollen which has a low linolenic acid content. The rationale behind this was to answer the question of whether lipid peroxidation is involved in the rapid viability loss and reduced membrane integrity of, in particular, the unsaturated-lipid pollen types. Viability and membrane integrity degraded more rapidly at 75% RH than at 40% RH. All pollen species showed deesterification of acyl chains of lipids but no detectable peroxidation at both RH levels. Considerable amounts of lipid-soluble antioxidants were detected that did not degrade during storage. Free fatty acids and lysophospholipids were formed during storage, the effects of which on membranes are discussed. These degradation products were very prominent in the short-lived Papaver pollen. The loss of viability does coincide with phospholipid deesterification. A significant decrease of the phospholipid content occurred at 75% RH, but not at 40% RH. Based on compositional analyses of phospholipids and newly formed free fatty acids, it was concluded that the deesterification of acyl chains from the lipids occurred at random. We suggest that, due to the low water content of the pollen, free radicals rather than unspecific acyl hydrolases are involved in the deesterification process.     

Analysis of composition and structure of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> membranes from wild-type and ethanol-adapted strains

Clostridium thermocellum is a candidate organism for consolidated bioprocessing of lignocellulosic biomass into ethanol. However, commercial use is limited due to growth inhibition at modest ethanol concentrations. Recently, an ethanol-adapted strain of C. thermocellum was produced. Since ethanol adaptation in microorganisms has been linked to modification of membrane lipids, we tested the hypothesis that ethanol adaptation in C. thermocellum involves lipid modification by comparing the fatty acid composition and membrane anisotropy of wild-type and ethanol-adapted strains. Derivatization to fatty acid methyl esters provided quantitative lipid analysis. Compared to wild-type, the ethanol-adapted strain had a larger percentage of fatty acids with chain lengths >16:0 and showed a significant increase in the percentage of 16:0 plasmalogens. Structural identification of fatty acids was confirmed through mass spectral fragmentation patterns of picolinyl esters. Ethanol adaptation did not involve modification at sites of methyl branching or the unsaturation index. Comparison of steady-state fluorescence anisotropy experiments, in the absence and presence of ethanol, provided evidence for the effects of ethanol on membrane fluidity. In the presence of ethanol, both strains displayed increased fluidity by approximately 12%. These data support the model that ethanol adaptation was the result of fatty acid changes that increased membrane rigidity that counter-acted the fluidizing effect of ethanol.     

Membrane lipid biosynthesis in Acholeplasma laidlawii B: Investigations into the in vivo regulation of the quantity and hydrocarbon chain lengths of de novo biosynthesized fatty acids in response to exogenously supplied fatty acids

The regulation of the nature and quantity of the fatty acids produced in vivo by Acholeplasma laidlawii B in the presence of various exogenous fatty acids has been investigated. In the presence of exogenous medium- or long-chain fatty acids, the organism appears to reduce the amounts of de novo biosynthesized fatty acids in its cellular lipid pool by two distinct mechanisms: an excretion of biosynthesized fatty acids to the growth medium as free fatty acids, and a reduction in total de novo biosynthetic output. These two mechanisms do not suffice to maintain constant total membrane lipid levels, but they do appear to significantly moderate the effect of exogenous fatty acids on the level of membrane lipid. In the presence of short-chain fatty acids, total membrane lipid levels are not elevated. Exogenous fatty acids can cause shifts in the average chain length of de novo biosynthesized fatty acids; the magnitudes and directions of these shifts can be correlated with the specificity of the exogenous species for esterification to the 1- or the 2-position of the glycerol moiety of membrane glycerolipids. As the various endogenously synthesized fatty acids differ in their positional specificity for glycerolipid esterification, we propose that the competition of an exogenous species with significant specificity for a particular position with the endogenously derived fatty acids specific for that position can selectively depress the synthesis of such endogenously derived species, thereby altering the overall product spectrum of de novo fatty acid biosynthesis in vivo.     

Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids

In the presence of sublethal concentrations of phenol, 4-chlorophenol, and p-cresol in the growth medium, cells of Escherichia coli modified the fatty acid composition of their lipids. The result of these changes was an increase in the degree of saturation of lipids probably in order to compensate an increase of fluidity of the membrane induced by the phenols. Supplementation of the growth medium with saturated fatty acids could also enhance the degree of lipid saturation due to the incorporation of the acyl chains in the phospholipids. At the same time the growth of cells was less inhibited than in unsupplemented cells. The increase of tolerance of cells by manipulating the lipid composition indicates that the membrane structure plays a crucial role in the mode of action of phenols.     

β-oxidation enzymes and the carnitine-dependent oxidation of palmitate and palmitoyl CoA in mitochondria from avocado

Two sites for the β-oxidation of fatty acids in avocado (Persea americana L.) mesocarp exist. One site is the microbody, the other the mitochondrion. It is apparent that the mitochondrial membrane barrier, which remains intact after sucrose density gradient centrifugation, prevents rapid access of acyl CoA substrates to matrix β-oxidation sites. Thus, intact mitochondria showed little β-oxidation enzyme activity. Rupturing of the mitochondrial membrane allowed rapid access of the acyl CoA substrates to matrix sites. Consequently, in ruptured mitochondria, high O₂-oxidation enzyme activities were measured. O₂ uptake studies further distinguished the two organellar sites of β-oxidation. During palmitoyl CoA oxidation, O₂ uptake was reduced by catalase and increased by KCN in the microbodies, whilst mitochondrial O₂ uptake was unaffected by catalase and reduced by KCN. This reflected the differing fates of FADH₂, produced during the first β-oxidation step, in the two organelles. In addition, only the mitochondrial β-oxidation of fatty acids was carnitine-dependent.     

Lipid- and temperature-dependent structural changes in Acholeplasma laidlawii cell membranes

The lipids in cell membranes of Acholeplasma laidlawii were enriched with different fatty acids selected to produce membranes showing molecular motion discontinuities at temperatures between 10 and 35 °C. Molecular motion in these membranes was probed by ESR after labelling with 12-nitroxide stearate, and structure in these membranes was examined by electron microscopy after freeze-etching.Freeze-etching and electron microscopy showed that under certain conditions the particles in the A. laidlawii membranes aggregated, resulting in particle-rich and particle-depleted regions in the cell membrane. Depending upon the lipid content of the membrane, this aggregation could begin at temperatures well above the ESR-determined discontinuity. Aggregation increased with decreasing temperature but was completed at or near the discontinuity. However, cell membranes grown and maintained well below their ESR-determined discontinuity did not show maximum particle aggregation until after they had been exposed to temperatures at or above the discontinuity.The results show that temperatures at or near a phase transition temperature can induce aggregation of the membrane particles. This suggests that temperature-induced changes in the lipid phase of a biological membrane can induce phase separations which affect the topography of associated proteins.     

The LpxL acyltransferase is required for normal growth and penta‐acylation of lipid A in Burkholderia cenocepacia

Lipid A anchors the lipopolysaccharide (LPS) to the outer membrane and is usually composed of a hexa‐acylated diglucosamine backbone. Burkholderia cenocepacia, an opportunistic pathogen, produces a mixture of tetra‐ and penta‐acylated lipid A. “Late” acyltransferases add secondary acyl chains to lipid A after the incorporation of four primary acyl chains to the diglucosamine backbone. Here, we report that B. cenocepacia has only one late acyltransferase, LpxL (BCAL0508), which adds a myristoyl chain to the 2′ position of lipid A resulting in penta‐acylated lipid A. We also identified PagL (BCAL0788), which acts as an outer membrane lipase by removing the primary β ‐hydroxymyristate (3‐OH‐C14:0) chain at the 3 position, leading to tetra‐acylated lipid A. Unlike PagL, LpxL depletion caused reduced cell growth and defects in cell morphology, both of which were suppressed by overexpressing the LPS flippase MsbA (BCAL2408), suggesting that lipid A molecules lacking the fifth acyl chain contributed by LpxL are not good substrates for the flippase. We also show that intracellular B. cenocepacia within macrophages produced more penta‐acylated lipid A, suggesting lipid A penta‐acylation in B. cenocepacia is required not only for bacterial growth and morphology but also for adaptation to intracellular lifestyle.     

Fatty acids covalently bound to erythrocyte proteins undergo a differential turnover in vivo

Recently, covalently bound fatty acids have been identified on a variety of proteins. Many of these acyl proteins are physiologically important, and the lipid modification often appears to be essential for their function. In this investigation mature erythrocytes have been used to study in detail the metabolic behavior of protein-bound fatty acids. Although deficient in protein synthesis, these cells are still able to covalently attach [3H]palmitic acid to proteins located at the plasma membrane and its associated cytoskeleton. Linkage analyses demonstrated that the labeled polypeptides contained ester- or thioester-bound fatty acids. The covalent binding of fatty acid was rapidly reversible. Half-lives of the protein-bound fatty acid molecules ranged from less than 30 min to more than 3 h. The deacylation reaction was not due to a chemically labile linkage of protein and fatty acid but appeared to be physiologically induced. Differences in the fatty acid turnover rates between the acyl proteins suggested an independent regulation of their lipid turnover. A number of proteins underwent dynamic fatty acid acylation, indicating that palmitylated proteins undergoing fatty acid turnover are not a rare exception.     

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.