Cholesterol-containing Lipid Membranes Research Articles

Role of Cholesterol in Interaction of Ionic Liquids with Model Lipid Membranes and Associated Permeability.

In this work, we explored how the amount of cholesterol in the lipid membrane composed of phosphatidylcholine (POPC) or phosphatidylglycerol (POPG) affects the interaction with 1-dodecyl-3-methylimidazolium bromide ([C12MIM]+Br-) ionic liquids using various biophysical techniques. On interacting with the membrane, [C12MIM]+Br- leads to enhanced membrane permeability and induces membrane fusion, leading to an increase in vesicle size. The 2H-based solid-state NMR investigations of cholesterol-containing lipid membranes reveal that [C12MIM]+Br- decreases the lipid chain order parameters and counteracts the lipid condensation effect of cholesterol to some extent. Therefore, as the amount of cholesterol in the membrane increases, the membrane effect of [C12MIM]+Br- decreases. The effect of [C12MIM]+Br- on the membrane properties is more pronounced for POPC compared to that of POPG membranes. This suggests a dependence of these effects on the electrostatic interactions, indicating that the influence of [C12MIM]+Br- varies based on the lipid composition. The findings suggest that the presence of cholesterol can modulate the effect of [C12MIM]+Br- on membrane properties, with variations observed between POPC and POPG membranes, highlighting the importance of lipid composition. In short, this study provides insights into the intricate interplay between cholesterol, the lipid membrane, and the ionic liquid [C12MIM]+Br-.

Influence of docosahexaenoic acid on the interfacial behavior of cholesterol-containing lipid membranes: Interactions with small amphiphiles and hydration properties

Cholesterol is known to significantly modify both the structural and the dynamical properties of lipid membranes. On one side, the presence of free cholesterol molecules has been determined to stiffen the membrane bilayer by stretching the hydrophobic tails. Additionally, recent experimental and computational findings have made evident the fact that cholesterol also alters the dynamics and the hydration properties of the polar head groups of DPPC model lipid membranes. In turn, we have recently shown that the Omega-3 fatty acid docosahexaenoic acid, DHA, counteracts the effect of cholesterol on DPPC membrane's mechanical properties by fluidizing the bilayer. However, such behavior represents in fact a global outcome dominated by the larger lipid hydrophobic tails that neither discriminates between the different parts of the membrane nor elucidates the effect on membrane hydration and binding properties. Thus, we now perform molecular dynamics simulations to scrutinize the influence of DHA on the interfacial behavior of cholesterol-containing lipid membranes by characterizing their hydration properties and their binding to amphiphiles. We find that while cholesterol destabilizes interactions with amphiphiles and slightly weakens the lipid's hydration layer, the incorporation of DHA practically restores the interfacial behavior of pure DPPC.

Membrane Interaction of Ibuprofen with Cholesterol-Containing Lipid Membranes.

Deciphering the membrane interaction of drug molecules is important for improving drug delivery, cellular uptake, and the understanding of side effects of a given drug molecule. For the anti-inflammatory drug ibuprofen, several studies reported contradictory results regarding the impact of ibuprofen on cholesterol-containing lipid membranes. Here, we investigated membrane localization and orientation as well as the influence of ibuprofen on membrane properties in POPC/cholesterol bilayers using solid-state NMR spectroscopy and other biophysical assays. The presence of ibuprofen disturbs the molecular order of phospholipids as shown by alterations of the 2H and 31P-NMR spectra of the lipids, but does not lead to an increased membrane permeability or changes of the phase state of the bilayer. 1H MAS NOESY NMR results demonstrate that ibuprofen adopts a mean position in the upper chain/glycerol region of the POPC membrane, oriented with its polar carbonyl group towards the aqueous phase. This membrane position is only marginally altered in the presence of cholesterol. A previously reported result that ibuprofen is expelled from the membrane interface in cholesterol-containing DMPC bilayers could not be confirmed.

Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR

The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, 15N NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2 is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical 19F-19F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15-20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels.

DOPAL derived alpha-synuclein oligomers impair synaptic vesicles physiological function

Parkinson’s disease is a neurodegenerative disorder characterized by the death of dopaminergic neurons and by accumulation of alpha-synuclein (aS) aggregates in the surviving neurons. The dopamine catabolite 3,4-dihydroxyphenylacetaldehyde (DOPAL) is a highly reactive and toxic molecule that leads to aS oligomerization by covalent modifications to lysine residues. Here we show that DOPAL-induced aS oligomer formation in neurons is associated with damage of synaptic vesicles, and with alterations in the synaptic vesicles pools. To investigate the molecular mechanism that leads to synaptic impairment, we first aimed to characterize the biochemical and biophysical properties of the aS-DOPAL oligomers; heterogeneous ensembles of macromolecules able to permeabilise cholesterol-containing lipid membranes. aS-DOPAL oligomers can induce dopamine leak in an in vitro model of synaptic vesicles and in cellular models. The dopamine released, after conversion to DOPAL in the cytoplasm, could trigger a noxious cycle that further fuels the formation of aS-DOPAL oligomers, inducing neurodegeneration.

Structure of Cholesterol in Lipid Rafts.

Rafts, or functional domains, are transient nano-or mesoscopic structures in the plasma membrane and are thought to be essential for many cellular processes such as signal transduction, adhesion, trafficking, and lipid or protein sorting. Observations of these membrane heterogeneities have proven challenging, as they are thought to be both small and short lived. With a combination of coarse-grained molecular dynamics simulations and neutron diffraction using deuterium labeled cholesterol molecules, we observe raftlike structures and determine the ordering of the cholesterol molecules in binary cholesterol-containing lipid membranes. From coarse-grained computer simulations, heterogenous membranes structures were observed and characterized as small, ordered domains. Neutron diffraction was used to study the lateral structure of the cholesterol molecules. We find pairs of strongly bound cholesterol molecules in the liquid-disordered phase, in accordance with the umbrella model. Bragg peaks corresponding to ordering of the cholesterol molecules in the raftlike structures were observed and indexed by two different structures: a monoclinic structure of ordered cholesterol pairs of alternating direction in equilibrium with cholesterol plaques, i.e., triclinic cholesterol bilayers.

How sterol tilt regulates properties and organization of lipid membranes and membrane insertions

Serving as a crucial component of mammalian cells, cholesterol critically regulates the functions of biomembranes. This review focuses on a specific property of cholesterol and other sterols: the tilt modulus χ that quantifies the energetic cost of tilting sterol molecules inside the lipid membrane. We show how χ is involved in determining properties of cholesterol-containing membranes, and detail a novel approach to quantify its value from atomistic molecular dynamics (MD) simulations. Specifically, we link χ with other structural, thermodynamic, and mechanical properties of cholesterol-containing lipid membranes, and delineate how this useful parameter can be obtained from the sterol tilt probability distributions derived from relatively small-scale unbiased MD simulations. We demonstrate how the tilt modulus quantitatively describes the aligning field that sterol molecules create inside the phospholipid bilayers, and we relate χ to the bending rigidity of the lipid bilayer through effective tilt and splay energy contributions to the elastic deformations. Moreover, we show how χ can conveniently characterize the “condensing effect” of cholesterol on phospholipids. Finally, we demonstrate the importance of this cholesterol aligning field to the proper folding and interactions of membrane peptides. Given the relative ease of obtaining the tilt modulus from atomistic simulations, we propose that χ can be routinely used to characterize the mechanical properties of sterol/lipid bilayers, and can also serve as a required fitting parameter in multi-scaled simulations of lipid membrane models to relate the different levels of coarse-grained details.

Structure-Function Relationships of Antimicrobial Piscidins 1 and 3 bound to Cholesterol-Containing Lipid Membranes

Isolated in the mast cells of hybrid-striped sea bass, piscidin 1 (p1) and piscidin 3 (p3) are antimicrobial, cationic, and amphipathic peptides that have demonstrated broad-spectrum activity against bacteria, fungi, viruses, and cancer cells. Both p1 and p3 adopt an alpha-helical structure when bound to phospholipid membranes. p1 is the more active of the two isoforms, exhibiting higher lytic activity on different bacterial strains, as well as erythrocytes. This research uses various biophysical methods to investigate the differences in the backbone structure and bilayer location of piscidin bound to different lipid bilayers of biological relevance.To mimic the composition of human erythrocytes, a mixture of 4:1 palmitoyl-oleoyl-phosphatidylcholine (POPC):cholesterol (CHL) at pH 7.4 was used. Oriented bilayer samples prepared with 1:40 peptide:lipid were analyzed using solid-state NMR yielding 15N-1H dipolar couplings as well as 15N amide chemical shifts. These data were used to compute a high-resolution atomic-level structure of p1 and p3 bound to zwitterionic bilayers. The peptide's backbone structure is largely conserved across various bacterial and mammalian membrane mimics, suggesting that the difference in a peptide's activity on various membranes is more reliant on the peptide's side-chain and its position within the membrane.The structural results were supplemented with dye leakage assays in order to probe the peptide's activity on lipid vesicles, mimicking bacterial and mammalian cells. These results lend insight into how specific changes in peptide's structure may result in varying activity on different membrane systems. Molecular dynamics (MD) simulations were used to predict the peptide's bilayer location and depth of insertion. Membrane thinning, an event known to precede pore formation, was shown using MD. Overall, these experiments help obtain principles to design novel antibiotic pharmaceuticals with low hemolytic effects and high lytic activity on bacteria.

Determining the Structures and Dynamics of Antimicrobial Piscidins 1 and 3 Bound to Cholesterol-Containing Lipid Membranes using Solid-State NMR Techniques

Isolated in the mast cells of hybrid-striped sea bass, piscidin 1 (p1) and piscidin 3 (p3) are antimicrobial, cationic, and amphipathic peptides that have demonstrated broad-spectrum activity against bacteria, and virally infected cells. Both p1 and p3 adopt an alpha-helical structure when bound to phospholipid membranes. Interestingly, p1 is the more active of the two isoforms, exhibiting higher lytic activity on different bacterial strains, as well as erythrocytes. This research uses solid-state NMR to investigate the differences in structure and dynamics of p1 and p3 bound to different lipid bilayers of biological relevance. Our current study aims at elucidating both the structure and dynamics of p1 and p3 in mammalian mimetic membranes containing cholesterol. Here, we use 4:1 palmitoyl-oleoyl-phosphatidylcholine (POPC):cholesterol (CHL) at a pH of 7.4, to closely mimic the composition of human erythrocytes at physiological pH. Oriented bilayer samples were prepared with 1:30 peptide:lipid and were analyzed using high-resolution solid-state NMR that yielded an 15N-1H dipolar coupling as well as 15N and 1H chemical shifts for the amide NH bond of each residue. Together, these data can be used to compute high-resolution atomic structures of the two isoforms when bound to the surface of the bilayer. Dynamics of p1 and p3 have also been investigated using one-dimensional 15N solid-state NMR. Differences in structure and dynamics for p1 and p3 when they are bound to mammalian and bacterial lipid systems provide the basis for identifying structural moieties of the peptides that are directly related to their respective activities. Enhancing our understanding of how these peptides interact with different lipid systems will aid in the design of novel antibiotic pharmaceuticals that exhibit low hemolytic effects, while maximizing inhibitory activity on bacteria.

Molecular organization of antifungal antibiotic amphotericin B in lipid monolayers studied by means of Fluorescence Lifetime Imaging Microscopy

Amphotericin B (AmB) is a life-saving polyene antibiotic used to treat deep-seated mycotic infections. Both the mode of therapeutic action as well as toxic side effects are directly dependent on molecular organization of the drug. Binding of AmB to lipid monolayers formed with dipalmitoylphosphatidylcholine, pure and containing 40 mol% cholesterol or ergosterol, the sterols of human and fungi respectively, has been examined by means of Fluorescence Lifetime Imaging Microscopy. AmB emits fluorescence with the characteristic lifetimes dependent on actual molecular organization: tau(M2) < or = 10 ps and tau(M1) = 0.35 ns in the monomeric state, the emission from the S(2) and the S(1) states respectively and tau(D) = 14 ns and tau(A) = 3.5 ns in the form of a dimer and associated dimers respectively. Analysis of the Langmuir-Blodgett films reveals that AmB binds to the lipid membranes and to the cholesterol-containing lipid membranes preferentially in the form of associated dimers. The same form of AmB appears in the membranes containing ergosterol but additionally the monomers and dimers of the drug can be observed, which can severely affect molecular organization of the lipid membrane. The results are discussed in terms of selectivity of AmB towards the ergosterol-containing biomembranes of fungi.

Structural investigations of pneumolysin/lipid complexes.

Pneumolysin, a virulence factor from the human pathogen Streptococcus pneumoniae, is a water-soluble protein which forms ring-shaped oligomeric structures upon binding to cholesterol-containing lipid membranes. It induces vesicle aggregation, membrane pore formation and withdrawal of lipid material into non-bilayer proteolipid complexes. Solid-state magic angle spinning and wideline static NMR, together with freeze-fracture electron microscopy, are used to characterize the phase changes in fully hydrated cholesterol-containing lipid membranes induced by the addition of pneumolysin. A structural model for the proteolipid complexes is proposed where a 30-50-meric pneumolysin ring lines the inside of a lipid torus. Cholesterol is found to be essential to the fusogenic action of pneumolysin.

Flip-Flop of Doxorubicin across Erythrocyte and Lipid Membranes

Doxorubicin, an anticancer drug, is extruded from multidrug resistant (MDR) cells and from the brain by P-glycoprotein located in the plasma membrane and the blood–brain barrier, respectively. MDR-type drugs are hydrophobic and, as such, enter cells by diffusion through the membrane without the requirement for a specific transporter. The apparent contradiction between the presumably free influx of MDR-type drugs into MDR cells and the efficient removal of the drugs by P-glycoprotein, an enzyme with a limited ATPase activity, prompted us to examine the mechanism of passive transport within the membrane. The kinetics of doxorubicin transport demonstrated the presence of two similar sized drug pools located in the two leaflets of the membrane. The transbilayer movement of doxorubicin occurred by a flip-flop mechanism of the drug between the two membrane leaflets. At 37°, the flip-flop exhibited a half-life of 0.7 min, in both erythrocyte membranes and cholesterol-containing lipid membranes. The flip-flop was inhibited by cholesterol and accelerated by high temperatures and the fluidizer benzyl alcohol. The rate of doxorubicin flux across membranes is determined by both the massive binding to the membranes and the slow flip-flop across the membrane. The long residence-time of the drug in the inner leaflet of the plasma membrane allows P-glycoprotein a better opportunity to remove it from the cell.