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.

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.

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.

O 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 (log < 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.

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.

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 or liver cells; and (3) 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.

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.

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.

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 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.

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.

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 Redβ 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.

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.

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.

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).

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.

The C-terminus of the phage λ Orf recombinase is involved in DNA binding.

Phage λ Orf substitutes for the activities of the Escherichia coli RecFOR proteins in vivo and is therefore implicated as a recombination mediator, encouraging the assembly of bacterial RecA onto single-stranded DNA (ssDNA) coated with SSB. Orf exists as a dimer in solution, associates with E. coli SSB and binds preferentially to ssDNA.

Acyl transfer from phosphocholine lipids to melittin.

Transfer of fatty acyl groups from membrane phospholipids to melittin, a commonly studied membrane-active peptide, has been observed to occur over extended time periods. Transfer can be detected after 1-2 days and selectively targets amino groups at the N-terminal end of the peptide.

Influence of lipids on the interfacial disposition of respiratory syncytical virus matrix protein.

The propensity of a matrix protein from an enveloped virus of the Mononegavirales family to associate with lipids representative of the viral envelope has been determined using label-free methods, including tensiometry and Brewster angle microscopy on lipid films at the air-water interface and atomic force microscopy on monolayers transferred to OTS-treated silicon wafers. This has enabled factors that influence the disposition of the protein with respect to the lipid interface to be characterized. In the absence of sphingomyelin, respiratory syncytial virus matrix protein penetrates monolayers composed of mixtures of phosphocholines with phosphoethanolamines or cholesterol at the air-water interface.

The synergistic action of melittin and phospholipase A2 with lipid membranes: development of linear dichroism for membrane-insertion kinetics.

Here we present data on the kinetics of insertion of melittin, a peptide from bee venom, into lipid membranes of different composition. Another component of bee venom is the enzyme phospholipase A2 (PLA₂). We have examined the interaction of melittin and PLA₂ with liposomes both separately and combined and demonstrate that they work synergistically to disrupt the membranes.

Free-energy relationships for the interactions of tryptophan with phosphocholines.

In membrane proteins and peptides, tryptophan exhibits a marked tendency to occur in locations that correspond to the interfacial region of the lipid bilayer. The relative contributions of electrostatic, dipolar, hydrophobic and conformational effects on the interactions of tryptophan with lipids have been the subject of much speculation. In order to elucidate the fundamental properties of tryptophan-phosphocholine interactions in the absence of competing factors such as protein conformation and membrane perturbation, we have determined the binding characteristics of a homologous series of tryptophan analogues to 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) in deuterochloroform using NMR titrimetric approaches.