Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

Synthesis of unsaturated fatty acids

This article reviews synthetic routes leading to polyunsaturated fatty acids having “skipped” double bonds. Emphasis is placed on the “acetylenic approach”.The suitability of building blocks, their condensation reactions as well as the controlled reduction of triple bonds to cis double bonds are discussed. In addition, the application of the various methods to the preparation of polyunsaturated fatty acids labelled with ³H and/or ¹⁴C at distinct positions of the molecules is reviewed.     

A synopsis of the process of lipid peroxidation since the discovery of the essential fatty acids

Eighty years ago, Burr and Burr, introduced for the first time the concept of essential fatty acids. Now is very well known that requirements for polyunsaturated fatty acids PUFAs can not be met by de novo metabolic processes within mammalian tissues. Animals are absolutely dependent on plants for providing the two major precursors of the n-6 and n-3 fatty acids, C18:2n-6; linoleic and C18:3n-3; α-linolenic acids. In animal tissues these precursors are transformed to fatty acids containing three to six double bonds. During the last four decades the interest in polyunsaturated fatty acids has augmented manifolds, and the number of published studies is rising each year. The current impetus for this interest has been mainly the observation that PUFAs and their metabolites have several physiological roles including: energy provision, membrane structure, cell signaling and regulation of gene expression. In addition the observation that PUFAs are targets of lipid peroxidation opens a new important area of investigation. Melatonin, the main secretory product of the pineal gland, efficiently scavenges both the hydroxyl and peroxyl radicals counteracting lipid peroxidation in biological membranes. In addition the two key pineal biochemical functions, lipoxygenation and melatonin synthesis may be synergistically regulated by the status of n-3 essential fatty acids. At the retina level, free radicals may preferentially react with the membrane polyunsaturated fatty acids leading to the release of lipoperoxide radicals. These lipoperoxides can induce oxidative stress linked to membrane lysis, damage to neuronal membranes may be related to alteration of visual function.     

Expression and Purification of Integral Membrane Fatty Acid Desaturases

Fatty acid desaturase enzymes perform dehydrogenation reactions leading to the insertion of double bonds in fatty acids, and are divided into soluble and integral membrane classes. Crystal structures of soluble desaturases are available; however, membrane desaturases have defied decades of efforts due largely to the difficulty of generating recombinant desaturase proteins for crystallographic analysis. Mortierella alpina is an oleaginous fungus which possesses eight membrane desaturases involved in the synthesis of saturated, monounsaturated and polyunsaturated fatty acids. Here, we describe the successful expression, purification and enzymatic assay of three M. alpina desaturases (FADS15, FADS12, and FADS9-I). Estimated yields of desaturases with purity >95% are approximately 3.5% (Ca. 4.6 mg/L of culture) for FADS15, 2.3% (Ca. 2.5 mg/L of culture) for FADS12 and 10.7% (Ca. 37.5 mg/L of culture) for FADS9-I. Successful expression of high amounts of recombinant proteins represents a critical step towards the structural elucidation of membrane fatty acid desaturases.     

Structure and dynamic properties of diunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine lipid bilayer from molecular dynamics simulation

Unsaturated fatty acid chains are known to be an essential structural part of biomembranes, but only monounsaturated chains have been included in the molecular dynamics (MD) simulations of membrane systems. Here we present a 1-ns MD simulation for a diunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLPC; 16:0/18:2[delta9,12]) bilayer. The structural behavior of the phosphatidylcholine headgroup, the glycerol backbone, and the hydrating water were assessed and found to be consistent with the existing information about similar systems from both experimental and computational studies. Further analysis was focused on the structure of the double bond region and the effects of the diunsaturation on the bilayer interior. The behavior of the diunsaturated sn-2 chains is affected by the tilted beginning of the chain and the four main conformations of the double bond region. The double bonds of the sn-2 chains also influenced the characteristics of the saturated chains in the sn-1 position. Furthermore, extreme conformations of the sn-2 chains existed that are likely to be related to the functional role of the double bonds. The results here point out the importance of polyunsaturation for the biological interpretations deduced from the membrane MD simulations.     

Phospholipid,ester-linked fatty acid profiles as reproducible assays for changes in prokaryotic community structure of estuarine sediments

Phospholipid, ester-linked fatty acid profiles showed changes in benthic prokaryotic community structure reflecting culture manipulations that were both quantitative and statistically significant. Fatty acid structures, including the position and cis/ trans geometry of double bonds, were chemically verified by GC/MS after appropriate derivatization. The fatty acid profiles of independent flasks showed reproducible shifts when manipulated identically and significant differences when manipulated with different treatments. The absence of polyunsaturated fatty acids indicated that the consortia were predominantly prokaryotic. The prokaryotic consortia of different treatments could be differentiated by the proportions of cyclopropyl fatty acids and the proportions and geometry of monounsaturated fatty acids.     

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis.

Acyl-lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl-lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step-wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri- and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.     

Interactions of phenytoin with rat brain synaptosomes examined by fluorescent fatty acid probes

Phenytoin (PHT) modified the fluorescent characteristics of anthroyloxy-fatty acids in synaptosomal membranes. Association of PHT with synaptosomal membranes caused the greatest change when the fluorescent probe was located at the 6-carbon position of N-(anthroyloxy)stearic acid and was incorporated into the membranes. Phenytoin and 6-(anthroyloxy)stearic acid compete for high affinity binding regions which are probably lipid domains within the membrane. Phenytoin has a weaker association with the sites than the fluorescent fatty acids. Divalent cations, e.g. Mg²⁺ or Ca²⁺, are required to observe maximal change in polarization of fluorescence of fatty acid probes in the presence of PHT. It is proposed that the membrane lipid bilayer reorganizes to accommodate exogenous compounds, such as phenytoin or the fatty acid probe in order to permit the most efficient packing of lipids. This reorganization of the lipid bilayer may influence membrane enzyme activities and ion channels.     

Polyunsaturated membranes are required for photosynthetic competence in a mutant of Arabidopsis

High levels of polyunsaturation are characteristic of all the membranes of plant and animal cells. For example, the chloroplasts of leaf cells contain about 75–80% polyunsaturated fatty acids. For the extra-chloroplast membranes in leaf cells and the membranes of non-photosynthetic tissues, values of 60–65% are typical. We report here the production of Arabidopsis double mutants that contain negligible levels of polyunsaturated fatty acids. The mutants were not capable of autotrophic growth and produced extremely chlorotic cotyledons and leaves. However, on sucrose media, the double mutants were robust plants showing strong leaf and root development. These observations indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids. By contrast, photosynthesis is one process that does require high levels of membrane polyunsaturation.     

Polyunsaturated fatty acids alter sterol transbilayer domains in LM fibroblast plasma membrane

Sterols are asymmetrically distributed between the leaflets of animal cell plasma membranes. Although transbilayer migration of sterols is extremely rapid, s to min, previous experimental manipulations have not altered their transmembrane steady-state distribution. However, the effect of polyunsaturated fatty acids has not been reported. When cultured in a lipid-free, chemically defined culture medium, LM fibroblasts do not synthesize polyunsaturated fatty acids but will incorporate polyunsaturated fatty acids into their plasma membranes if supplied in the medium. Sterol transbilayer distribution in LM plasma membranes was determined from quenching of fluorescence of dehydroergosterol by trinitrophenyl groups selectively attached to the exofacial leaflet. When cells are cultured in lipid-free media, 28.1% of the plasma membrane sterol is located in the exofacial (outside) leaflet. In contrast, when cells are cultured with linoleate- or linolenate-supplemented medium, 71.8% and 75.5% of the plasma membrane sterol is exofacial, respectively.     

Selective internalization of arachidonic acid by endothelial cells.

The asymmetric distribution of phospholipids in bovine endothelial-cell membranes was probed with 2,4,6-trinitrobenzenesulphonate and purified phospholipase A2. The data suggest that phosphotidylethanolamine is primarily located in the inner lipid bilayer, as reported for other cell types. Stearic acid is taken up by the endothelial cells and is randomly distributed among the membrane phospholipids. In contrast, the polyunsaturated fatty acids (arachidonic, eicosatrienoic and eicosapentaenoic acids) have initial incorporation into the phosphatidylcholine fraction. These fatty acids then undergo a time-dependent transfer from phosphatidylcholine to phosphatidylethanolamine. Thus we propose that endothelial cells possess a mechanism for the selective internalization of polyunsaturated fatty acids.     

Evidence for a mechanism by which omega-3 polyunsaturated lipids may affect membrane protein function

We have calculated the lateral pressure profile from well-converged, experimentally validated, molecular dynamics simulations of hydrated lipid bilayer membranes containing highly polyunsaturated fatty acids. The three simulations, each 30 ns in length, contain omega-3 fatty acids, omega-6 fatty acids, and a mixture of omega-3 fatty acids and cholesterol and were continued from previously published simulations that demonstrated excellent agreement with a wide variety of experimental measurements. We find that the distribution of lateral stress within the hydrophobic core of the membrane is sensitively dependent on the degree of chain unsaturation and on the presence of cholesterol. Replacing omega-3 fatty acids with omega-6 chains, or incorporating cholesterol into the membrane, shifts the repulsive lateral chain pressure away from the lipid/water interface toward the bilayer interior. This may support a previously proposed mechanism by which lipid composition may affect conformational equilibrium for integral membrane proteins.     

Phospholipids and their fatty acids in mitochondria, synaptosomes and myelin from the liver and brain of trout and rat: a new view on the role of fatty acids in membranes

The content of different phospholipids (PL) and their fatty acid (FA) composition in subcellular fractions from the liver and brain of rat (Rattus rattus) and trout (Salmo irideus) were estimated. It was shown that despite higher content of unsaturated fatty acids in myelin compared to synaptosomes, the unsaturation index of the latter is equal or higher than that of myelin. The total content of PL polyunsaturated fatty acids (PUFA) was shown to be higher in membrane structures with more active ion transport (mitochondria). This feature seemed to be characteristic of membranes from both representatives of homoiotherms and poikilotherms studied. A possible role for PUFA information within the lipid monolayer of areas with different capacity to accept electrons and transport them along a sort of intermolecular 'tunnel' is discussed. The double bonds of PUFA in this area seem to be able to produce bonds similar to conjugated bonds.     

Homogeneous catalytic hydrogenation of lipids in the photosynthetic membrane: Effects on membrane structure and photosynthetic activity

We have carried out a series of experiments in which the lipid composition of the photosynthetic membrane has been altered by the homogeneous catalytic hydrogenation of the unsaturated fatty acid residues of membrane lipids. The modified membrane was investigated by electron microscopy, electron-spin resonance and fluorescence polarization methods. Alteration in the functional characteristics of the hydrogenated membrane was monitored by the measurement of photophosphorylation and electron-transport activities. The following results were found. (a) Saturation of 10% of the fatty acyl double bonds induced a definite decrease in the dimension of both thylakoids and loculi. Microdensitometry showed that these structural changes arose from a thickening of the single membranes with a simultaneous decrease in the spacing between membranes. These changes might be accounted for by the alignment of the hydrocarbon chains of saturated lipids and the increased hydrophobicity of the membranes. (b) The orientational pattern of chlorophyll-a molecules was not altered by saturating up to 50% of fatty acyl double bonds in membrane lipids, indicating that the energy-transfer processes amongst the chlorophyll molecules remained functional after hydrogenation. (c) Saturation of double bonds of lipids inhibited whole electron transport prior to the inhibition of Photosystem II and Photosystem I activity, which may suggest that the unsaturation level of fatty acids plays a crucial role by ensuring the lateral mobility of plastoquinone between Photosystem II and Photosystem I.     

Effects of long-chain cis-unsaturated fatty acids and their alcohol analogs on aggregation of bovine platelets and their relation with membrane fluidity change

The effects of long-chain cis-unsaturated fatty acids with different alkyl chain lengths and different numbers of double bonds on aggregation of bovine platelets and membrane fluidity were investigated. All the cis-unsaturated fatty acids tested inhibited aggregation and at the same time increased membrane fluidity in accordance with their inhibitory effects. The saturated fatty acids and trans-unsaturated fatty acid tested for comparison had much lower or no effects on aggregation and membrane fluidity. The inhibitory effects of mono cis-unsaturated fatty acids increased with increase of their alkyl chain length. cis-Unsaturated fatty acids with two or more double bonds had more inhibitory effects than mono-unsaturated fatty acids. The position of the double bonds had less influence than the number of double bonds. We also examined the effects of cis-unsaturated fatty acids on membrane fluidity with diphenylhexatriene and anthroyloxy derivatives of fatty acids as probes and observed increased fluidity to be considerable in the membrane. The alcohol analogs of cis-unsaturated fatty acids also inhibited aggregation and increased membrane perturbation. These results suggest that the inhibition of platelet aggregation by cis-unsaturated compounds is due to perturbation of the lipid layer.     

Chain ordering in liquid crystals: II. Structure of bilayer membranes

The ordering of the hydrocarbon chain interior of bilayer membranes has been calculated using the molecular field approximation developed in previous work on liquid crystals. Different statistical averages are evaluated by exact summation over all conformations of a single chain in the field due to neighboring molecules. The internal energy of each conformation, as well as contributions arising from interaction with the molecular field and from a lateral pressure on the chain have been included.The results describe properties of both lipid monolayers and bilayers. For monolayers, the calculated pressure-area relationships are in good agreement with experimental observations. The order parameter for hydrocarbon chains in bilayers (or monolayers) as a function of temperature, lateral pressure and position along the chain, is shown and compared with the available NMR data. Combining the results of calculation and NMR measurements we obtain the value for intrinsic lateral pressure within bilayer membranes, in excellent agreement with direct measurements on surface monolayers.The calculation also gives average length of hydrocarbon chains, thermal expansion coefficient and fraction of bonds in gauche conformations. The effect of cholesterol and proteins within the bilayer is qualitatively described, and the contribution of the bilayer interior to membrane elasticity is determined.     

Mechanism of Long-Chain Free Fatty Acid Protonation at the Membrane-Water Interface

Long-chain free fatty acids (FFAs) play an important role in several physiological and pathological processes such as lipid fusion, adjustments of membrane permeability and fluidity, and the regulation of enzyme and protein activities. FFA-facilitated membrane proton transport (flip-flop) and FFA-dependent proton transport by membrane proteins (e.g., mitochondrial uncoupling proteins) are governed by the difference between FFA’s intrinsic pK_a value and the pH in the immediate membrane vicinity. Thus far, a quantitative understanding of the process has been hampered, because the pK_a value shifts upon moving the FFA from the aqueous solution into the membrane. For the same FFA, pK_a values between 5 and 10.5 were reported. Here, we systematically evaluated the dependence of pK_a values on chain length and number of double bonds by measuring the ζ-potential of liposomes reconstituted with FFA at different pH values. The experimentally obtained intrinsic pK_a values (6.25, 6.93, and 7.28 for DOPC membranes) increased with FFA chain length (C16, C18, and C20), indicating that the hydrophobic energy of transfer into the bilayer is an important pK_a determinant. The observed pK_a decrease in DOPC with increasing number of FFA double bonds (7.28, 6.49, 6.16, and 6.13 for C20:0, C20:1, C20:2, and C20:4, respectively) is in line with a decrease in transfer energy. Molecular dynamic simulations revealed that the ionized carboxylic group of the FFAs occupied a fixed position in the bilayer independent of chain length, underlining the importance of Born energy. We conclude that pK_a is determined by the interplay between the energetic costs for 1) burying the charged moiety into the lipid bilayer and 2) transferring the hydrophobic protonated FFA into the bilayer.     

Interaction modes of long-chain fatty acids in dipalmitoylphosphatidylcholine bilayer membrane: contrast to mode of inhalation anesthetics

The effects of long-chain fatty acids (four saturated and two unsaturated fatty acids, one derivative) on phase transitions of dipalmitoylphosphatidylcholine (DPPC) bilayer membranes were examined in the low concentration region, and the results were compared with those for an inhalation anesthetic. The effects of all fatty acids on the pre- and main-transition temperatures of the DPPC bilayer membrane appeared in the concentration range of μM order while that of the anesthetic appeared in the mM order. The appearance modes of these ligand actions were significantly different from one another. The three differential partition coefficients of the ligands between two phases of the DPPC bilayer membrane were evaluated by applying the thermodynamic equation to the variation of the phase-transition temperatures. The DPPC bilayer membranes showed the different receptivity for the ligands; the saturated fatty acids had an affinity for gel phase whereas unsaturated fatty acids and an anesthetic had an affinity for liquid-crystalline phase to the contrary. In particular, the receptivity for the ligands in the gel phase markedly changed depending on kinds of ligands. The interaction modes between the DPPC and fatty acid molecules in the gel phase were considered from the hexagonal lattice model. The disappearance compositions of the pretransition by the fatty acids coincided with the compositions at which the membrane is all covered by the units in each of which two fatty acids molecules are regularly distributed in the hexagonal lattice in a different way, and the distribution depended on the chain length and existence of a double bond for the fatty acids. The interpretation did not hold for the case of the anesthetic at all, which proved that a number of anesthetic molecules act the surface region of the bilayer membrane nonspecifically. The present study clearly implies that DPPC bilayer membranes have high ability to recognize kinds of ligand molecules and can discriminate among them with specific interaction by the membrane states.