The Chemical Reactivity of Membrane Lipids.

It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.

The association of lipids with amyloid fibrils.

Amyloid formation continues to be a widely studied area because of its association with numerous diseases, such as Alzheimer's and Parkinson's diseases. Despite a large body of work on protein aggregation and fibril formation, there are still significant gaps in our understanding of the factors that differentiate toxic amyloid formation in vivo from alternative misfolding pathways. In addition to proteins, amyloid fibrils are often associated in their cellular context with several types of molecule, including carbohydrates, polyanions, and lipids. This review focuses in particular on evidence for the presence of lipids in amyloid fibrils and the routes by which those lipids may become incorporated. Chemical analyses of fibril composition, combined with studies to probe the lipid distribution around fibrils, provide evidence that in some cases, lipids have a strong association with fibrils. In addition, amyloid fibrils formed in the presence of lipids have distinct morphologies and material properties. It is argued that lipids are an integral part of many amyloid deposits in vivo, where their presence has the potential to influence the nucleation, morphology, and mechanical properties of fibrils. The role of lipids in these structures is therefore worthy of further study.

Peptide lipidation in lysophospholipid micelles and lysophospholipid-enriched membranes.

Acyl transfer from lipids to membrane-associated peptides is a well-documented process, leading to the generation of a lipidated peptide and a lysolipid. In this article, we demonstrate that acyl transfer from lysophosphatidylcholines (lysoPCs) to the peptide melittin also occurs, both in micelles of pure lysolipid and in lipid/lysolipid mixtures. In the case of bilayers containing lysolipids, acyl transfer from the lysolipid is marginally favoured over transfer from the lipid. In pure bilayers of saturated lipids, the introduction of even small amounts of lysolipid appears to significantly increase the reactivity towards lipidation.

17O NMR spectroscopy as a tool to study hydrogen bonding of cholesterol in lipid bilayers.

Cholesterol is a crucial component of biological membranes and can interact with other membrane components through hydrogen bonding. NMR spectroscopy has been used previously to investigate this bonding, however this study represents the first 17O NMR spectroscopy study of isotopically enriched cholesterol. We demonstrate the 17O chemical shift is dependent on hydrogen bonding, providing a novel method for the study of cholesterol in bilayers.

Lysis of membrane lipids promoted by small organic molecules: Reactivity depends on structure but not lipophilicity.

Several organic molecules of low molecular weight (<150 Da) are demonstrated to have substantial membrane-lytic potential despite having a low predicted lipophilicity (logD < 1 at neutral pH). In aqueous liposome dispersions, 38 aromatic compounds were tested for their ability to either promote lipid hydrolysis or directly participate in chemical reactions with lipid molecules. Behaviors observed included acyl transfer from the lipid to form a lipidated compound, both with and without concomitant lysolipid formation; increases in the rate of lipid hydrolysis without lipidation; and no reactivity. The variation in activity, including a notably higher activity for heterocycles such as amino-substituted benzimidazoles and indazoles, demonstrates the potential to predict or "design-in" lytic activity once the rules that govern reactivity are better understood. The nature of this chemical instability has significant ramifications for the use or presence of lipids in diverse fields such as materials chemistry, food chemistry, and cell physiology.

Far from Inert: Membrane Lipids Possess Intrinsic Reactivity That Has Consequences for Cell Biology.

In this article, it is hypothesized that a fundamental chemical reactivity exists between some non-lipid constituents of cellular membranes and ester-based lipids, the significance of which is not generally recognized. Many peptides and smaller organic molecules have now been shown to undergo lipidation reactions in model membranes in circumstances where direct reaction with the lipid is the only viable route for acyl transfer. Crucially, drugs like propranolol are lipidated in vivo with product profiles that are comparable to those produced in vitro. Some compounds have also been found to promote lipid hydrolysis. Drugs with high lytic activity in vivo tend to have higher toxicity in vitro. Deacylases and lipases are proposed as key enzymes that protect cells against the effects of intrinsic lipidation. The toxic effects of intrinsic lipidation are hypothesized to include a route by which nucleation can occur during the formation of amyloid fibrils.

Lytic reactions of drugs with lipid membranes.

Propranolol is shown to undergo lipidation reactions in three types of lipid membrane: (1) synthetic single-component glycerophospholipid liposomes; (2) liposomes formed from complex lipid mixtures extracted from E. coli or liver cells; and (3) in cellulo in Hep G2 cells. Fourteen different lipidated propranolol homologues were identified in extracts from Hep G2 cells cultured in a medium supplemented with propranolol. This isolation of lipidated drug molecules from liver cells demonstrates a new drug reactivity in living systems. Acyl transfer from lipids to the alcoholic group of propranolol was favoured over transfer to the secondary amine. Migration of acyl groups from the alcohol to the amine was diminished. Other drugs that were examined did not form detectable levels of lipidation products, but many of these drugs did affect the lysolipid levels in model membranes. The propensity for a compound to induce lysolipid formation in a model system was found to be a predictor for phospholipidosis activity in cellulo.

The influence of cholesterol on melittin lipidation in neutral membranes.

The effects of cholesterol on the process of intrinsic lipidation, whereby an acyl chain is transferred from a lipid as donor to a membrane-associated acceptor molecule, have been explored using melittin as the acceptor. Membranes comprising lipids with saturated acyl chains (1,2-dipalmitoyl sn-glycero-3-phosphocholine, DPPC; 1,2-dimyristoyl sn-glycero-3-phosphocholine, DMPC) yielded no acyl transfer, whereas membranes composed of lipids with unsaturated acyl chains (1,2-dioleoyl sn-glycero-3-phosphocholine, DOPC; 1-palmitoyl-2-oleoyl sn-glycero-3-phosphocholine, POPC) produced detectable lipidation activity. For all lipids, inclusion of cholesterol led to a significant increase in lipidation activity, with the greatest effect observed for 20 mol% cholesterol in POPC. In the case of membranes composed of POPC, the inclusion of cholesterol also produced small changes in the selectivity for transfer from the sn-1 vs. sn-2 positions of the lipid. Qualitatively, for fluid membranes, the trend in lipidation activity exhibits a positive correlation with the bending modulus of the bilayer and is accounted for in terms of the penetration depth of the peptide. Access of water to reactive intermediates also has the potential to influence lipidation rates.

Modulation of Backbone Flexibility for Effective Dissociation of Antibacterial and Hemolytic Activity in Cyclic Peptides.

Bacterial resistance to antibiotic therapy is on the rise and threatens to evolve into a worldwide emergency: alternative solutions to current therapies are urgently needed. Cationic amphipathic peptides are potent membrane-active agents that hold promise as the next-generation therapy for multidrug-resistant infections. The peptides' behavior upon encountering the bacterial cell wall is crucial, and much effort has been dedicated to the investigation and optimization of this amphipathicity-driven interaction. In this study we examined the interaction of a novel series of nine-membered flexible cyclic AMPs with liposomes mimicking the characteristics of bacterial membranes. Employed techniques included circular dichroism and marker release assays, as well as microbiological experiments. Our analysis was aimed at correlating ring flexibility with their antimicrobial, hemolytic, and membrane activity. By doing so, we obtained useful insights to guide the optimization of cyclic antimicrobial peptides via modulation of their backbone flexibility without loss of activity.

The lipidation profile of aquaporin-0 correlates with the acyl composition of phosphoethanolamine lipids in lens membranes.

The lens fiber major intrinsic protein (otherwise known as aquaporin-0 (AQP0), MIP26 and MP26) has been examined by mass spectrometry (MS) in order to determine the speciation of acyl modifications to the side chains of lysine residues and the N-terminal amino group. The speciation of acyl modifications to the side chain of one specific, highly conserved lysine residue (K238) and the N-terminal amino group of human and bovine AQP0 revealed, in decreasing order of abundance, oleoyl, palmitoyl, stearoyl, eicosenoyl, dihomo-γ-linolenoyl, palmitoleoyl and eicosadienoyl modifications. In the case of human AQP0, an arachidonoyl modification was also found at the N-terminus. The relative abundances of these modifications mirror the fatty acid composition of lens phosphatidylethanolamine lipids. This lipid class would be expected to be concentrated in the inner leaflet of the lens fiber membrane to which each of the potential AQP0 lipidation sites is proximal. Our data evidence a broad lipidation profile that is both species and site independent, suggesting a chemical-based ester aminolysis mechanism to explain such modifications.

Optimised conditions for the synthesis of (17)O and (18)O labelled cholesterol.

Conditions are described for the preparation of cholesterol with (17)O and (18)O labels from i-cholesteryl methyl ether using minimal amounts of isotopically enriched water. Optimum yields employed trifluoromethanesulfonic acid as catalyst in 1,4-dioxane at room temperature with 5 equivalents of water. An isotopic enrichment >90% of that of the water used for the reaction could be attained. Tetrafluoroboric acid could also be used as catalyst, at the expense of a lower overall reaction yield. Byproducts from the reaction included dicholesteryl ether, methyl cholesteryl ether, compounds formed by ether hydrolysis, and olefins arising from elimination reactions. Reactions in tetrahydrofuran yielded significant amounts of cholesteryl ethers formed by reaction with alcohols arising from hydrolysis of the solvent.

The association of defensin HNP-2 with negatively charged membranes: A combined fluorescence and linear dichroism study.

The association of defensin HNP-2 with negatively charged membranes has been studied using a new approach that combines fluorescence and linear dichroism (LD) spectroscopies with simulated LD spectra in order to characterise the binding kinetics and bound configurations of the peptide. Binding to membranes composed of mixtures of diacylglycerophosphocholines (PC) with either diacylglycerophosphoglycerol (PG) or diacylglycerophosphoserine (PS) was conducted at lipid:peptide ratios that yielded binding, but not membrane fusion. HNP-2 association with membranes under these conditions was a 2 stage-process, with both stages exhibiting first order kinetics. The fast initial step, with a half-life of < 1 min, was followed by a slower step with a half-life of > 3 min. Conversion between the states was estimated to have an enthalpy of activation of approximately 10 kJ mol(-1) and an entropy of activation of -0.2 kJ K mol(-1). LD spectra corresponding to each of the membrane bound states were generated by non-linear regression using a standard kinetic model. These spectra are interpreted in comparison with spectra calculated using the program Dichrocalc and reveal that the peptide associates with membranes in a small number of stable configurations. All of these configurations have a significant proportion of ß-sheet structure residing in the plane of the membrane. Two configurations support structures previously proposed for defensins in membranes.

Phage ORF family recombinases: conservation of activities and involvement of the central channel in DNA binding.

Genetic and biochemical evidence suggests that λ Orf is a recombination mediator, promoting nucleation of either bacterial RecA or phage Redß recombinases onto single-stranded DNA (ssDNA) bound by SSB protein. We have identified a diverse family of Orf proteins that includes representatives implicated in DNA base flipping and those fused to an HNH endonuclease domain. To confirm a functional relationship with the Orf family, a distantly-related homolog, YbcN, from Escherichia coli cryptic prophage DLP12 was purified and characterized. As with its λ relative, YbcN showed a preference for binding ssDNA over duplex. Neither Orf nor YbcN displayed a significant preference for duplex DNA containing mismatches or 1-3 nucleotide bulges. YbcN also bound E. coli SSB, although unlike Orf, it failed to associate with an SSB mutant lacking the flexible C-terminal tail involved in coordinating heterologous protein-protein interactions. Residues conserved in the Orf family that flank the central cavity in the λ Orf crystal structure were targeted for mutagenesis to help determine the mode of DNA binding. Several of these mutant proteins showed significant defects in DNA binding consistent with the central aperture being important for substrate recognition. The widespread conservation of Orf-like proteins highlights the importance of targeting SSB coated ssDNA during lambdoid phage recombination.

Acyl transfer from membrane lipids to peptides is a generic process.

The generality of acyl transfer from phospholipids to membrane-active peptides has been probed using liquid chromatography-mass spectrometry analysis of peptide-lipid mixtures. The peptides examined include melittin, magainin II, PGLa, LAK1, LAK3 and penetratin. Peptides were added to liposomes with membrane lipid compositions ranging from pure phosphatidylcholine (PC) to mixtures of PC with phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol. Experiments were typically conducted at pH7.4 at modest salt concentrations (90 mM NaCl). In favorable cases, lipidated peptides were further characterized by tandem mass spectrometry methods to determine the sites of acylation. Melittin and magainin II were the most reactive peptides, with significant acyl transfer detected under all conditions and membrane compositions. Both peptides were lipidated at the N-terminus by transfer from PC, phosphatidylethanolamine, phosphatidylserine or phosphatidylglycerol, as well as at internal sites: lysine for melittin; serine and lysine for magainin II. Acyl transfer could be detected within 3h of melittin addition to negatively charged membranes. The other peptides were less reactive, but for each peptide, acylation was found to occur in at least one of the conditions examined. The data demonstrate that acyl transfer is a generic process for peptides bound to membranes composed of diacylglycerophospholipids. Phospholipid membranes cannot therefore be considered as chemically inert toward peptides and by extension proteins.

A chiral probe for the acute phase proteins alpha-1-acid glycoprotein and alpha-1-antitrypsin based on europium luminescence.

Reversible and selective binding of a dynamically racemic europium(III) complex to α(1)-acid glycoprotein and α(1)-antitrypsin is characterised by a significant change in the europium total emission spectral fingerprint and the switching on of a large circularly polarised luminescence (CPL) signal from the metal centre. Observation of an induced CD into the ligand chromophore in the presence of α(1)-AGP allows a structure for the protein-bound complex to be postulated. A direct determination of elevated α(1)-AGP levels in human serum was achieved by monitoring changes in the intensity ratio of Eu emission bands.

Resolving the kinetics of lipid, protein and peptide diffusion in membranes.

Recent developments in the understanding of molecular diffusion phenomena in membranes are reviewed. Both model bilayers and biological membranes are considered in respect of lateral diffusion, rotational diffusion and transverse diffusion (flip-flop). For model systems, particular attention is paid to recent data obtained using surface-specific techniques such as sum frequency generation vibrational spectroscopy on supported lipid bilayers, and fluorescence correlation spectroscopy on giant unilamellar vesicles, both of which have yielded new insights into the intrinsic rates of diffusion and the energetic barriers to processes such as lipid flip-flop. Advances in single-molecule and many-molecule fluorescence methodologies have enabled the observation of processes such as anomalous diffusion for some membrane species in biological membranes. These are discussed in terms of new models for the role of membrane interactions with the cytoskeleton, the effects of molecular crowding in membranes, and the formation of lipid rafts. The diffusion of peptides, proteins and lipids is considered, particularly in relation to the means by which antimicrobial peptide activity may be rationalized in terms of membrane poration and lipid flip-flop.

The innate reactivity of a membrane associated peptide towards lipids: acyl transfer to melittin without enzyme catalysis.

The innate reactivity of the peptide melittin (H-GIGAVLKVLTTGLPALISWIKRKRQQ-NH(2)) towards membrane lipids has been explored using LC-MS methods. The high sensitivity afforded by LC-MS analysis enabled acyl transfer to the peptide to be detected, within 4 h, from membranes composed of phosphocholines (PCs). Acyl transfer from PCs was also observed from mixtures of PC with phosphoserine (PS) or phosphoglycerol (PG). In the latter case, transfer from PG was also detected. The half-lives for melittin conversion varied between 24 h and 75 h, being fastest for POPC and slowest for DOPC/DMPG mixtures. The order of reactivity for amino groups on the peptide was N-terminus > K23 ≫ K21 > K7. Products arising from double-acylation of melittin were detected as minor components, together with a putative component derived from transesterification involving S18 of the peptide.

The reproducibility of phospholipid analyses by MALDI-MSMS.

The identification of phosphocholine and phosphoethanolamine lipids by MALDI TOF/TOF, including characterisation of the headgroup and delineation of the acyl chain at each position of the glycerol backbone, has been explored using lipids representative of each type. The relative intensities of fragments involving the neutral loss of one or other of the acyl chains from ion adducts of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine (OPPC) were compared. For POPC and POPE, a statistical preference for the loss of the chain from the sn-1 position was observed in the presence of lithium. For OPPC this selectivity was reversed for one of the fragments. In the absence of lithium, fragmentation was favoured at the sn-2 position for all lipids. In all cases, spectra obtained in the presence of lithium yielded more intense product ion peaks. Although Collision Induced Dissociation (CID) could be used for complete lipid characterisation, LIFT™ was found to be a better method due to the presence of a greater number of distinguishing product ion peaks and a better shot-to-shot reproducibility of peak intensities.