Is the cholesterol bilayer domain a barrier to oxygen transport into the eye lens?
ABSTRACT: In the eye lens, the oxygen partial pressure is very low and the cholesterol (Chol) content in cell membranes is very high. Disturbance of these quantities results in cataract development. In human lens membranes, both bulk phospholipid-Chol domains and the pure Chol bilayer domains (CBDs) were experimentally detected. It is hypothesized that the CBD constitutes a significant barrier to oxygen transport into the lens. Transmembrane profiles of the oxygen diffusion-concentration product, obtained with electron paramagnetic resonance spin-labeling methods, allow evaluation of the oxygen permeability (PM) of phospholipid membranes but not the CBD. Molecular dynamics simulation can independently provide components of the product across any bilayer domain, thus allowing evaluation of the PM across the CBD. Therefore, to test the hypothesis, MD simulation was used. Three bilayers containing palmitoyl-oleoyl-phosphorylcholine (POPC) and Chol were built. The pure Chol bilayer modeled the CBD, the 1:1 POPC-Chol bilayer modeled the bulk membrane in which the CBD is embedded, and the POPC bilayer was a reference. To each model, 200 oxygen molecules were added. After equilibration, the oxygen concentration and diffusion profiles were calculated for each model and multiplied by each other. From the respective product profiles, the PM of each bilayer was calculated. Favorable comparison with experimental data available only for the POPC and POPC-Chol bilayers validated these bilayer models and allowed the conclusion that oxygen permeation across the CBD is ~10 smaller than across the bulk membrane, supporting the hypothesis that the CBD is a barrier to oxygen transport into the eye lens.
Project description:Molecular dynamics (MD) simulations of a mono-cis-unsaturated 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayer and a POPC bilayer containing 50mol% cholesterol (POPC-Chol50) were carried out for 200ns to compare the spatial organizations of the pure POPC bilayer and the POPC bilayer saturated with Chol. The results presented here indicate that saturation with Chol significantly narrows the distribution of vertical positions of the center-of-mass of POPC molecules and POPC atoms in the bilayer. In the POPC-Chol50 bilayer, the same moieties of the lipid molecules are better aligned at a given bilayer depth, forming the following clearly separated membrane regions: the polar headgroup, the rigid core consisting of steroid rings and upper fragments of the acyl chains, and the fluid hydrocarbon core consisting of Chol chains and the lower fragments of POPC chains. The membrane surface of the POPC-Chol50 bilayer is smooth. The results have biological significance because the POPC-Chol50 bilayer models the bulk phospholipid portion of the fiber-cell membrane in the eye lens. It is hypothesized that in the eye lens cholesterol-induced smoothing of the membrane surface decreases light-scattering and helps to maintain lens transparency.
Project description:Models created with molecular dynamics simulations are used to compare the organization and dynamics of cholesterol (Chol) molecules in three different environments: (1) a hydrated pure Chol bilayer that models the Chol bilayer domain, which is a pure Chol domain embedded in the bulk membrane; (2) a 2-palmitoyl-3-oleoyl-d-glycerol-1-phosphorylcholine bilayer saturated with cholesterol (POPC-Chol50) that models the bulk membrane; (3) a Chol crystal. The computer model of the hydrated pure Chol bilayer is stable on the microsecond time scale. Some structural characteristics of Chol molecules in the Chol bilayer are similar to those in the POPC-Chol50 bilayer (e.g., tilt of Chol rings and chains), while others are similar to those in Chol crystals (e.g., surface area per Chol, bilayer thickness). The key result of this study is that the Chol bilayer has, unexpectedly, a dynamic structure, with Chol mobility similar to that in the POPC-Chol50 bilayer though slower. This is the major difference compared to Chol crystals, where Chol molecules are immobile. Also, water accessibility to Chol-OH groups in the Chol bilayer is not limited. On average, each Chol molecule makes 2.3 hydrogen bonds with water in the Chol bilayer, compared with 1.7 hydrogen bonds in the POPC-Col50 bilayer.
Project description:Understanding the toxicity of ionic liquids (ILs) is crucial in the search of greener chemicals. By comparing in vivo toxicity and in vitro interactions determined between compounds and biomimetic lipid membranes, more detailed toxicity vs. structure relation can be obtained. However, determining the interactions between non-surface-active compounds and liposomes has been a challenging task. Organisational changes induced by ILs and IL-like spirocyclic compounds within 1,6-diphenyl-1,3,5-hexatriene-doped biomimetic liposomes was studied by steady-state fluorescence anisotropy technique. The extent of organisational changes detected within the liposome bilayers were compared to the toxicity of the compounds determined using Vibrio Fischeri bacteria. Four liposome compositions made of pure 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocoline (POPC) and mixtures of POPC, 1-palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS), and cholesterol (Chol) were tested as biomimetic models. Changes observed within the POPC/POPS/Chol 55:20:25 bilayers correlated the best with the toxicity results: ten out of twelve compounds followed the trend of increasing bilayer disorder - increasing toxicity. The study suggests that the toxicity of non-surface-active compounds is dependent on their ability to diffuse into the bilayers. The extent of bilayer's organisational changes correlates rather well with the toxicity of the compounds. Highly sensitive technique, such as fluorescence anisotropy measurements, is needed for detecting subtle changes within the bilayer structures.
Project description:Cytolytic protein (Cyt) is a member of insecticidal proteins produced by Bacillus thuringiensis. Cyt protein has activity against insect cells and mammalian cells, which differ in lipid and cholesterol composition. This study presents the lipid binding behavior of Cyt2Aa2 protein on model membranes containing different levels of cholesterol content by combining Quartz Crystal Microbalance with Dissipation (QCM-D) and Atomic Force Microscopy (AFM). QCM-D results revealed that cholesterol enhances the binding rate of Cyt2Aa2 protein onto lipid bilayers. In addition, the thicker lipid bilayer was observed for the highest cholesterol content. These results were confirmed by AFM. The analysis of protein surface coverage as a function of time showed a slower process for 5:0 and 5:0.2 (POPC:Chol) ratios than for 5:1 and 5:2 (POPC:Chol) ratios. Significantly, the Cyt2Aa2-lipid binding behavior and the protein?lipid layer were different for the 5:3 (POPC:Chol) ratio. Furthermore, AFM images revealed a transformation of Cyt2Aa2/lipid layer structure from strip pattern to ring shape structures (which showed a strong repulsion with AFM tip). In summary, cholesterol increases the binding rate and alters the lipid binding behavior of Cyt2Aa2 protein, although it is not required for Cyt2Aa2 protein binding onto lipid bilayers.
Project description:Human lens lipid membranes prepared using a rapid solvent exchange method from the total lipids extracted from the clear lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across coexisting membrane domains. Membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape and also indicate a high fluidity and hydrophobicity of the membrane center. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane. Properties of human membranes are compared with those obtained for membranes made of lipids extracted from cortex and nucleus of porcine and bovine eye lenses.
Project description:Cytolytic toxin (Cyt) is a toxin among Bacillus thuringiensis insecticidal proteins. Cyt toxin directly interacts with membrane lipids for cytolytic action. However, low hemolytic activity is desired to avoid non-specific effects in mammals. In this work, the interaction between Cyt2Aa2 toxin and model lipid bilayers mimicking the erythrocyte membrane was investigated for Cyt2Aa2 wild type (WT) and the T144A mutant, a variant with lower hemolytic activity. Quartz crystal microbalance with dissipation (QCM-D) results revealed a smaller lipid binding capacity for the T144A mutant than for the WT. In particular, the T144A mutant was unable to bind to the phosphatidylcholine lipid (POPC) bilayer. However, the addition of cholesterol (Chol) or sphingomyelin (SM) to the POPC bilayer promoted binding of the T144 mutant. Moreover, atomic force microscopy (AFM) images unveiled small aggregates of the T144A mutant on the 1:1 sphingomyelin/POPC bilayers. In contrast, the lipid binding trend for WT and T144A mutant was comparable for the 1:0.4 POPC/cholesterol and the 1:1:1 sphingomyelin/POPC/cholesterol bilayers. Furthermore, the binding of WT and T144A mutant onto erythrocyte cells was investigated. The experiments showed that the T144A mutant and the WT bind onto different areas of the erythrocyte membrane. Overall the results suggest that the T144 residue plays an important role for lipid binding.
Project description:A distinguishing feature of Archaeal plasma membranes is that their phospholipids contain ether-links, as opposed to bacterial and eukaryotic plasma membranes where phospholipids primarily contain ester-links. Experiments show that this chemical difference in headgroup-tail linkage does produce distinct differences in model bilayer properties. Here we examine the effects of salt on bilayer structure in the case of an ether-linked lipid bilayer. We use molecular dynamics simulations and compare equilibrium properties of two model lipid bilayers in NaCl salt solution - POPC and its ether-linked analog that we refer to as HOPC. We make the following key observations. The headgroup region of HOPC "adsorbs" fewer ions compared to the headgroup region of POPC. Consistent with this, we note that the Debye screening length in the HOPC system is ? 10% shorter than that in the POPC system. Herein, we introduce a protocol to identify the lipid-water interfacial boundary that reproduces the bulk salt distribution consistent with Gouy-Chapman theory. We also note that the HOPC bilayer has excess solvent in the headgroup region when compared to POPC, coinciding with a trough in the electrostatic potential. Waters in this region have longer autocorrelation times and smaller lateral diffusion rates compared to the corresponding region in the POPC bilayer, suggesting that the waters in HOPC are more strongly coordinated to the lipid headgroups. Furthermore, we note that it is this region of tightly coordinated waters in the HOPC system that has a lower density of Na+ ions. Based on these observations we conclude that an ether-linked lipid bilayer has a lower binding affinity for Na+ compared to an ester-linked lipid bilayer.
Project description:The translocation of lipids across membranes (flip-flop) is an important biological process. Slow exchange on a physiological timescale allows the creation of asymmetric distributions of lipids across cellular membranes. The location of lipids and their rate of exchange have important biological consequences, especially for lipids involved in cellular signaling. We investigated the translocation of cholesterol, ceramide, and diacylglycerol in two model bilayers using molecular dynamics simulations. We estimate half times for flip-flop for cholesterol, diacylglycerol, and ceramide of 20 ?s, 30 ?s, and 10 ms in a POPC bilayer, compared with approximately 30 min, 30 ms, and 30 s in a model raft bilayer (1:1:1 PSM, POPC, and cholesterol). Cholesterol has a large (54 kJ/mol) free energy of exchange between the POPC and raft bilayer, and therefore, it strongly prefers the more ordered and rigid raft bilayer over the more liquid POPC bilayer. Ceramide and diacylglycerol have relatively small free energies of exchange, suggesting nearly equal preference for both bilayers. This unexpected result may have implications for ceramide and diacylglycerol signaling and membrane localization.
Project description:Gramicidin A (gA) is a 15-amino-acid antibiotic peptide with an alternating L-D sequence, which forms (dimeric) bilayer-spanning, monovalent cation channels in biological membranes and synthetic bilayers. We performed molecular dynamics simulations of gA dimers and monomers in all-atom, explicit dilauroylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), and 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers. The variation in acyl chain length among these different phospholipids provides a way to alter gA-bilayer interactions by varying the bilayer hydrophobic thickness, and to determine the influence of hydrophobic mismatch on the structure and dynamics of both gA channels (and monomeric subunits) and the host bilayers. The simulations show that the channel structure varied little with changes in hydrophobic mismatch, and that the lipid bilayer adapts to the bilayer-spanning channel to minimize the exposure of hydrophobic residues. The bilayer thickness, however, did not vary monotonically as a function of radial distance from the channel. In all simulations, there was an initial decrease in thickness within 4-5 Å from the channel, which was followed by an increase in DOPC and POPC or a further decrease in DLPC and DMPC bilayers. The bilayer thickness varied little in the monomer simulations-except one of three independent simulations for DMPC and all three DLPC simulations, where the bilayer thinned to allow a single subunit to form a bilayer-spanning water-permeable pore. The radial dependence of local lipid area and bilayer compressibility is also nonmonotonic in the first shell around gA dimers due to gA-phospholipid interactions and the hydrophobic mismatch. Order parameters, acyl chain dynamics, and diffusion constants also differ between the lipids in the first shell and the bulk. The lipid behaviors in the first shell around gA dimers are more complex than predicted from a simple mismatch model, which has implications for understanding the energetics of membrane protein-lipid interactions.
Project description:Interleukin-8alpha (IL-8alpha) is an antimicrobial peptide derived from the chemokine IL-8. Solution NMR was used to determine the atomic-resolution structure of IL-8alpha in SDS micelles. Solid-state NMR and tryptophan fluorescence were used to probe the interaction of IL-8alpha with model membranes. The peptide interacted differently with anionic versus purely zwitterionic micelles or bilayers. Tryptophan fluorescence demonstrated a deeper position of Trp4 in SDS micelles and POPC/POPG bilayers compared to pure POPC bilayers, consistent with (2)H order parameters, which also indicated a deeper position of the peptide in POPC/POPG bilayers compared to POPC bilayers. Paramagnetic probe data showed that IL-8alpha was situated roughly parallel to the SDS micelle surface, with a slight tilt that positioned the N-terminus more deeply in the micelle compared to the C-terminus. (15)N solid-state NMR spectra indicated a similar, nearly parallel position for the peptide in POPC/POPG bilayers. (31)P and (2)H solid-state NMR demonstrated that the peptide did not induce the formation of any nonlamellar phases and did not significantly disrupt bilayer orientation in aligned model membranes composed of POPC or POPC and POPG.