Structure Of Biological Membranes Research Articles

ChemInform Abstract: Polyelectrolyte-Sensitized Phospholipid Vesicles

Control of the structure and permeability of biological membranes is one of the most basic and least understood problems in contemporary biophysics. Diverse cellular processes require exquisite control of the membrane structure, including endocytosis and exocytosis, maintenance of the Golgi apparatus and the endoplasmic reticulum, and mitosis and meiosis, to mention just a few. Recently, significant progress has been made in controlling the permeability, and often the structures, of synthetic bilayer vesicles. These studies are especially important in that they help elucidate previously unimagined mechanisms for membrane control. Although no single model system is likely to utilize all of the rich capabilities exploited in nature, the synthetic systems can serve to identify concepts and mechanisms likely to be useful in a biological context. In addition, control of vesicular membrane systems may usher in novel chemistries for use in imaging, sensing, and therapeutic applications.

Molecular Dynamics Simulations of Mixed Acidic/Zwitterionic Phospholipid Bilayers

Anionic lipids are key components in the cell membranes. Many cell-regulatory and signaling mechanisms depend upon a complicated interplay between them and membrane-bound proteins. Phospholipid bilayers are commonly used as model systems in experimental or theoretical studies to gain insight into the structure and dynamics of biological membranes. We report here 200-ns-long MD simulations of pure (DMPC and DMPG) and mixed equimolar (DMPC/DMPG, DMPC/DMPS, and DMPC/DMPA) bilayers that each contain 256 lipids. The intra- and intermolecular interaction patterns in pure and mixed bilayers are analyzed and compared. The effect of monovalent ions (Na+) on the formation of salt-bridges is investigated. In particular, the number of Na+-mediated clusters in the presence of DMPS is higher than with DMPG and DMPA. We observe a preferential clustering of DMPS (and to some extent DMPA) lipids together rather than with DMPC molecules, which can explain the phase separation observed experimentally for DMPC/DMPS and DMPC/DMPA bilayers.

Haematological and Antioxidant Properties of Alloxan-Induced Diabetes Rats Supplemented with Antioxidant Micronutrients

Oxidative stress is elevated in patients with diabetes mellitus and oxidative modification of red cell membrane increases its fragility. Micronutrients with antioxidant effect protect structure and function of biological membranes. In the current study, manganese, copper and zinc, were supplemented in alloxan-induced diabetic rats for a period of 4 weeks. Haematological indices were determined by standard methods and results analyzed using InStat3 Statistical Software. Haemoglobin, packed cell volume, and red blood cell concentrations were 16.91±0.23g/dl, 50.0±1.11% and 5.63±0.32(1012/L) in controls 12.43±0.71g/dl, 36.42±1.38% and 4.58±0.23(1012/L) in treated unsupplemented group, and 13.84±0.43g/dl, 42.14±1.67% and 5.54±0.32(1012/L) in treated and supplemented groups respectively. Mean cell volume, mean cell haematocrit and mean cell haematocrit concentration values were 9.39±0.25fl, 3.15±0.12pg and 0.33±0.01g/L in controls, 8.18±0.46fl, 2.74±0.09pg and 0.34±0.62g/L in the treated unsupplemented group, and 7.99±0.65fl, 2.57±0.18pg and 0.32±0.01g/L in the treated and supplemented group respectively. Activities of superoxide dismutase, glutathione peroxidase and catalase were 2.00±0.17U/mg protein, 46.43±4.25U/mg protein and 59.86±1.08U/mg protein in the controls, 1.67±0.22U/mg protein, 36.88±3.91U/mg protein and 39.86±3.03U/mg protein in the unsupplemented group and 1.61±0.18U/mg protein, 44.86±1.82U/mg protein and 57.14±2.48U/mg protein in the supplemented group respectively. The concentration of malondialdehyde was 1.83±0.16nmol/ml in controls, 2.37±0.19nmol/ml in supplemented group and 1.91±1.14nmol/ml in the supplemented group. It may be concluded from the present study that antioxidant micronutrient improve some haematological and antioxidant indices and reduce lipid peroxidation in alloxan-induced diabetic rats. Keywords: diabetes, haematological indices, micronutrients, lipid peroxidation.

ChemInform Abstract: Synthesis of a Small Library of Mixed-Acid Phospholipids from D-Mannitol as a Homochiral Starting Material.

Phospholipids constitute the main structural component of cell membranes. Due to their amphiphilic nature, they associate into aggregate structures such as vesicles, micelles and bilayers. In biological phospholipid assemblies, the nature of the lipid components affects the function and dynamics of the membrane. 1) At the same time, since their discovery, liposomes have been widely employed as models of biological membranes. Especially, this membrane model is studied for application for drug delivery system. Therefore, current investigation for the structure of biological membranes and liposomes requires efficient methods for preparation of phospholipids. 2) Phospholipids with defined fatty acid composition 3) are widely used as components of artificial membranes for physiochemical studies. 4) Phospholipids with defined fatty acid composition are widely used as fluorescent, 5) radiolabelled, 6) and spin-labeled probes 7) for study of membrane motion and as photoactivatable probes for investigation of protein‐lipid interaction. 8) Phospholipids with defined fatty acid composition are also involved in signal transduction in the enzymatic processing of phospholipid. 9) A commonly employed method for synthesis involves specific deacylation of phospholipase A2 and reacylation of the resulting 2lysophospholipids with the desired acid. The drawback of this strategy is the difficulty for the preparation of large scale of desired compound. Therefore, efficient synthetic methods for the preparation of mixed diacyl phospholipids have to be developed. Here, we wish to report the synthesis of a small library of mixed diacyl phospholipids (Chart 1) starting from D-mannitol as the chiral starting material.

Changes in the structure of model biological membranes in the presence of perfluorooctanesulphonic acid – Electrochemical and EC-STM study

The changes caused by the presence of perfluorooctanesulphonic acid (PFOS) in the structure and electrochemical characteristics of model biological membranes composed of 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) supported on gold electrodes were examined using electrochemical techniques and electrochemical scanning tunneling microscopy. Phospholipid mono and bilayers were prepared using the Langmuir–Blodgett (LB) technique alone or combined with of the Langmuir–Schaefer (LB/LS) techniques. Square wave voltammetry was employed to monitor the incorporation of PFOS into already existing model membranes. The accessibility of potassium ferrocyanides to the electrode surface depended on the time of contact of the bilayer with PFOS solutions. Electrostatic attraction between PFOS present in the model membrane and positively charged probes resulted in the adsorption of the probe on the lipid membrane modified electrode. Even highly soluble species, i.e. hexaamineruthenium complex revealed adsorption on the gold electrodes modified with DMPC bilayers containing PFOS. The presence of PFOS in model biological membranes was visualized by means of electrochemical scanning tunneling microscopy (EC-STM). Topographically higher PFOS-enriched domains were clearly distinguishable among lower pure DMPC regions.

Self-Organization of the Escherichia Coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy

The Escherichia coli chemotaxis network is a model system for signal transduction and processing. Chemotaxis receptors assemble into large clusters containing tens of thousands of proteins which have been observed at cell poles and future division sites. Despite extensive study, it remains unclear how chemotaxis clusters form, what controls cluster size and density, and how the cellular location of clusters is robustly maintained in growing and dividing cells. Here, we use a super-resolution optical technique called photoactivated localization microscopy (PALM) to map the cellular locations of three proteins central to bacterial chemotaxis (the Tar receptor, CheY, and CheW) with a precision of 15 nm. We find that cluster sizes are approximately exponentially distributed, with no characteristic cluster size. One-third of receptors are part of smaller lateral clusters that have not been previously observed. Analysis of the relative cellular locations of 1.1 million individual proteins (from 326 cells) suggests that clusters form via stochastic self-assembly. The super-resolution PALM maps of E. coli receptors support a growing collection of evidence that stochastic self-assembly can create and maintain periodic structures in biological membranes, without direct cytoskeletal involvement or active transport.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Molecular Dynamics Simulations of In-Plane Density Fluctuations in Phospholipid Bilayers

At room temperature many lipids form the bilayer structure of biological membranes, which resembles liquid crystals. The structural features of the fluid lipid bilayer are well-known, but many details of the dynamic behavior of biological membranes still remain to be understood.Molecular motions in liquids can be monitored by the intermediate scattering function, F(q,t). In membranes it probes the propagation and decay of in-plane density flutuations at wave vector q. An attractive property of the intermediate scattering function is that it can equally well be determined from scattering experiments as from molecular dynamics simulations, opening the possibility of direct comparison between experimental and simulation data. Density fluctuations in lipid bilayers can stay correlated for hundreds of nanoseconds which implies that in contrast to simple liquids, an exponential decay of F(q,t) as suggested by a purely single diffusive model, does not describe how fluid membranes behave. Microsecond molecular dynamics simulations on thousands of lipids, both atomistic and coarse-grained, has been used to explore F(q,t). The atomistic simulations span membrane patches of tens of nanometers while the coarse-grained simulations, including almost 100 000 lipids, reach the low micrometer domain. The main purpose was to establish a dispersion relation for the density fluctuations, i.e. a relation between the wave vector and the decay rate. Different model functions are compared to find the dispersion relation that best fits the simulation data. The important question of how material properties (bending modulus and area compressibility) are related to the molecular structure of the complex liquid is adressed in the context of the different models.

Simulations of Lipid Bilayer Domain Formation: Effects of Steroid Structure and Asymmetry

There is a growing amount of evidence that laterally segregated domains of lipids are an integral part of biological membrane structure and function. Using Coarse Grain and Atomistic Molecular Dynamics Simulations we investigate the role of steroid structure and asymmetry in domain formation. Cholesterol, an essential component of animal membranes, appears to be well suited for ordering neighboring lipid chains and promoting domain formation. We demonstrate that alterations to the steroid headgroup hydrophobicity trigger a conversion from domain promoting to domain inhibiting. Those steroids which inhibit domain formation are observed to be less stable in the typical, upright orientation, and instead insert into the bilayer hydrophobic core and reside in an orientation perpendicular to the bilayer normal axis. A second set of simulations are used to address the role of bilayer asymmetry in domain formation. Cellular membranes are thought to be asymmetric, containing different lipid compositions on opposing leaflets, with the outer leaflet capable of domain formation and the inner leaflet uniformly disordered. These simulations suggest how domain formation is affected by an opposing uniformly disordered leaflet.

Water solutions of phenosan potassium salt: Influence on biological membrane structure and conductivity

Water solutions of phenosan potassium salt: Influence on biological membrane structure and conductivity

Self-Organization of the Escherichia coli Chemotaxis Network Imaged with Super-Resolution Light Microscopy

Author SummaryCells arrange their components—proteins, lipids, and nucleic acids—in organized and reproducible ways to optimize the activities of these components and, therefore, to improve cell efficiency and survival. Eukaryotic cells have a complex arrangement of subcellular structures such as membrane-bound organelles and cytoskeletal transport systems. However, subcellular organization is also important in prokaryotic cells, including rod-shaped bacteria such as E. coli, most of which lack such well-developed systems of organelles and motor proteins for transporting cellular cargoes. In fact, it has remained somewhat mysterious how bacteria are able to organize and spatially segregate their interiors. The E. coli chemotaxis network, a system important for the bacterial response to environmental cues, is one of the best-understood biological signal transduction pathways and serves as a useful model for studying bacterial spatial organization because its components display a nonrandom, periodic distribution in mature cells. Chemotaxis receptors aggregate and cluster into large sensory complexes that localize to the poles of bacteria. To understand how these clusters form and what controls their size and density, we use ultrahigh-resolution light microscopy, called photoactivated localization microscopy (PALM), to visualize individual chemoreceptors in single E. coli cells. From these high-resolution images, we determined that receptors are not actively distributed or attached to specific locations in cells. Instead, we show that random receptor diffusion and receptor–receptor interactions are sufficient to generate the observed complex, ordered pattern. This simple mechanism, termed stochastic self-assembly, may prove to be widespread in both prokaryotic and eukaryotic cells.

Lipid Gymnastics: Evidence of Complete Acyl Chain Reversal in Oxidized Phospholipids from Molecular Simulations

In oxidative environments, biomembranes contain oxidized lipids with short, polar acyl chains. Two stable lipid oxidation products are PoxnoPC and PazePC. PoxnoPC has a carbonyl group, and PazePC has an anionic carboxyl group pendant at the end of the short, oxidized acyl chain. We have used MD simulations to explore the possibility of complete chain reversal in OXPLs in POPC-OXPL mixtures. The polar AZ chain of PazePC undergoes chain reversal without compromising the lipid bilayer integrity at concentrations up to 25% OXPL, and the carboxyl group points into the aqueous phase. Counterintuitively, the perturbation of overall membrane structural and dynamic properties is stronger for PoxnoPC than for PazePC. This is because of the overall condensing and ordering effect of sodium ions bound strongly to the lipids in the PazePC simulations. The reorientation of AZ chain is similar for two different lipid force fields. This work provides the first molecular evidence of the “extended lipid conformation” in phospholipid membranes. The chain reversal of PazePC lipids decorates the membrane interface with reactive, negatively charged functional groups. Such chain reversal is likely to exert a profound influence on the structure and dynamics of biological membranes, and on membrane-associated biological processes.

Exogenously applied polyamines increase drought tolerance of rice by improving leaf water status, photosynthesis and membrane properties

Drought stress hampers rice performance principally by disrupting the plant–water relations and structure of biological membranes. This study appraised the role of polyamines (PAs) in improving drought tolerance in fine grain aromatic rice (Oryza sativa L.). Three PAs [putrescine (Put), spermidine (Spd) and spermine (Spm)] were used each at 10 μM as seed priming (by soaking seeds in solution) and foliar spray. Primed and non-primed seeds were sown in plastic pots with normal irrigation in a phytotron. At four-leaf stage, plants were subjected to drought stress by bringing the soil moisture down to 50% of field capacity by halting water supply. For foliar application, 10 μM solutions each of Put, Spd and Spm were sprayed at five-leaf stage. Results revealed that drought stress severely reduced the rice fresh and dry weights, while PAs application improved net photosynthesis, water use efficiency, leaf water status, production of free proline, anthocyanins and soluble phenolics and improved membrane properties. PAs improved drought tolerance in terms of dry matter yield and net photosynthesis was associated with the maintenance of leaf water status and improved water use efficiency. Among the antioxidants, catalase activity was negatively related to H2O2 and membrane permeability, which indicated alleviation of oxidative damage on cellular membranes by PAs application. Foliar application was more effective than the seed priming, and among the PAs, Spm was the most effective in improving drought tolerance.

Effects Of Hydration On The Dynamics Of Water In Lipid Bilayer Systems: A Molecular Dynamics Study

The properties of interlamellar water are critically important to the structure and function of biological membranes. Recent developments in femtosecond infrared spectroscopy on membrane systems at various hydration levels have opened the possibility of direct comparison between experiment and molecular dynamics simulations on the same time scales. The interpretation of experimental findings can benefit from a detailed computational study of water solvation structure and dynamics of inter-lamellar water at these hydration levels. In this presentation, we report molecular dynamics simulations of 1-palmitoyl-2-oleoyl-phosphatidylcholine POPC bilayers in the liquid-crystalline state and at three hydration levels. Simulations were performed in the canonical ensemble using the GROMACS software package. The extent to which water is influenced by the presence of membrane depends on the hydration level. We found the anisotropic diffusion constant of lipid water exhibits interesting crossover behavior as the water molecule moves from the head group region toward the bulk region. The anisotropic hydrodynamic diffusion of water is explained by structural perturbation of the water hydrogen bond network by the lipid. Radial distribution function, spatial distribution function, and power spectra of water are calculated to consolidate our interpretation. This work was partially supported by the National Science Foundation under Grant No. DGE-0221680.

Schiff Base Formation Between The Cholesterol Oxidation Product 3β-hydroxy-5-oxo-5,6-secocholestan-6-al And Amino Phospholipids

The keto-aldehyde 3β-hydroxy-5-oxo-5,6- secocholestan-6-al is formed by the oxidation of cholesterol with ozone. This oxidized form of cholesterol is associated with a number of pathological conditions including atherosclerotic plaques, Alzheimer's and Parkinson's diseases. We have shown earlier that the compound can react covalently with the amino group of phosphatidylethanolamine to form a Schiff base . Here, using a spectroscopic technique, we determine the kinetics of the Schiff base formation between 3β-hydroxy-5-oxo-5,6- secocholestan-6-al and dimyristoylphosphatidylethanolamine in both the gel and liquid crystalline states of the phospholipid. The activation energies of this reaction in the two states are also calculated. In addition, we determine that a Schiff base can also be formed with the amino group of phosphatidylserine, albeit with slower kinetics. These findings are significant as they show that oxidized cholesterol can react covalently not only with the amino groups of proteins, but also with the amino groups of phospholipids, potentially influencing the structure of biological membranes.

Membrane structure and the activity of phospholipase and sphingomyelinase D

Lipid-modifying enzymes play a vital role in the regulation of lipids as mediators of cell function. One example is the hydrolysis of phospholipids through phospholipase D (PLD), which produces the signalling molecule phosphatidic acid (PA). These processes at lipid membranes can be observed in situ through the application of different biophysical techniques. Thus, the hydrolysis of phosphatidylcholines by PLD was investigated, showing that the enzyme is highly affected in its catalytic activity by the lipid membrane structure. Briefly, by using Langmuir monolayers as a model system, we revealed that PLD activity depends on the segregation of the hydrolysis product PA within the monolayer. Hence, we could describe how the structure of the PA-rich domains is decisive for the activation and inhibition of PLD. This study demonstrates how membrane structure influences the activity of PLD and regulates the concentration of the lipid messenger PA.The current research project is aiming at describing a toxic component of the venom of brown spiders (Loxosceles), which has a rare enzymatic activity termed sphingomyelinase D (SMD). SMD catalyzes the conversion of sphingomyelin (SM) into ceramide-1-phosphate (Cer-1-P). While the enzymatic substrate SM is an integral constituent of many cell membranes, especially in the vascular epithelium and red blood cells, the reaction product Cer-1-P occurs in very low concentrations. Cer-1-P is suggested to be a novel lipid second messenger in cellular signal transduction events. At present, the precise mechanism of venom action is incompletely understood, but preliminary results show the strong effect of SMD activity on the membrane structure of giant unilamellar vesicles.In summary, the presented work depicts the correlation between membrane structures and the activity of lipid-modifying enzymes. This implements new models for the regulation of cellular processes through distinct structures of biological membranes.

Fabrication of Synaptotagmin II-Functionalized Neuromimetic Copolymeric Nanomembranes

Nanoscale copolymer membranes that mimic the innate structure and properties of biological lipid membranes possessing hydrophilic and hydrophobic elements to support protein folding were used for a fundamental examination of protein—polymer integration. This study has integrated the neural synaptotagmin II (Syt II) protein, a documented target of the hemagglutinin-33 (Hn-33) protein associated with botulinum neurotoxin type A during the infection process, into polymethyloxazoline—polydimethylsiloxane—polymethyloxazoline nanomembranes. By integrating Syt II into block copolymer membranes, we have developed a neural mimetic membrane toward Hn-33 targeting the applications in nanomaterial-mediated detection. This technology can serve as a robust stand-alone platform for toxin diagnostic studies, or as a coating for integration with micro-/nanofabricated devices and electrodes for protein—protein interaction-based detection. To assess enhanced membrane complexity and toxin specificity, studies assessing the co-insertion of trisialoganglioside-GT1b (GT1b) and Syt II into the nanomembranes were used as a subsequent platform for botulinum neurotoxin type B detection. Protein—membrane integration was confirmed with atomic force microscopy imaging, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Langmuir isotherm analysis.

Synaptic vesicle cycle

Neurotransmitter release from presynaptic nerve endings is mediated by Ca2+-dependent exocytosis of synaptic vesicles. During the past 15 years, major progress has been made in unravelling the molecular mechanisms underlying exocytosis and the recycling of synaptic vesicles. Exocytotic membrane fusion is mediated by the SNARE proteins synaptobrevin/VAMP, syntaxin 1, and SNAP-25, which are the only substrates of the Tetanus and Botulinum neurotoxin proteases. Upon membrane contact, the vesicular SNARE synaptobrevin forms complexes with the plasma membrane-resident SNAREs SNAP-25 and syntaxin 1. Complex formation proceeds from the N-terminal end towards the C-terminal membrane anchors, thus pulling the membranes together and initiating fusion (“zipper” hypothesis of SNARE function). In our own work, we have focussed on understanding the mechanisms of SNARE zippering in more detail using biochemical approaches and in-vitro fusion reactions with native and artificial membranes. Furthermore, we have studied the role of endosomes in synaptic vesicles and have performed a quantitative analysis of purified synaptic vesicles. The resulting molecular model yields novel insights into the structure of biological membranes, sheds light on neurotransmitter uptake and storage by synaptic vesicles, and provides a starting point for quantitative molecular models of vesicle docking and fusion.

Cytometry of raft and caveola membrane microdomains: From flow and imaging techniques to high throughput screening assays

The evolutionarily developed microdomain structure of biological membranes has gained more and more attention in the past decade. The caveolin-free "membrane rafts," the caveolin-expressing rafts (caveolae), as well as other membrane microdomains seem to play an essential role in controlling and coordinating cell-surface molecular recognition, internalization/endocytosis of the bound molecules or pathogenic organisms and in regulation of transmembrane signal transduction processes. Therefore, in many research fields (e.g. neurobiology and immunology), there is an ongoing need to understand the nature of these microdomains and to quantitatively characterize their lipid and protein composition under various physiological and pathological conditions. Flow and image cytometry offer many sophisticated and routine tools to study these questions. In this review, we give an overview of the past efforts to detect and characterize these membrane microdomains by the use of classical cytometric technologies, and finally we will discuss the results and perspectives of a new line of raft cytometry, the "high throughput screening assays of membrane microdomains," based on "lipidomic" and "proteomic" approaches.

Protein area occupancy at the center of the red blood cell membrane

In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least approximately 23% protein and less than approximately 77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than approximately 20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.

Using Compression Isotherms of Phospholipid Monolayers To Explore Critical Phenomena. A Biophysical Chemistry Experiment

Two experiments are described in which students explore phase transitions and critical phenomena by obtaining compression isotherms of phospholipid monolayers using a Langmuir trough. Through relatively simple analysis of their data students gain a better understanding of compression isotherms, the application of the Clapeyron equation, the balance between enthalpic and entropic contributions to the chemical potential, critical phenomena, and phase diagrams in general. Students use their data to determine the latent heat of transition, the entropy of transition, and the critical temperature for the liquid condensed to liquid expanded phase transition of a monolayer. Students also gain a general understanding of how molecular level changes in the structure of phospholipids, such as changes in chain length affect the structure and function of biological membranes.