S-layer-supported lipid membranes

Many prokaryotic organisms (archaea and bacteria) are covered by a regularly ordered surface layer (S-layer) as the outermost cell wall component. S-layers are built up of a single protein or glycoprotein species and represent the simplest biological membrane developed during evolution. Pores in S-layers are of regular size and morphology, and functional groups on the protein lattice are aligned in well-defined positions and orientations. Due to the high degree of structural regularity S-layers represent unique systems for studying the structure, morphogenesis, and function of layered supramolecular assemblies. Isolated S-layer subunits of numerous organisms are able to assemble into monomolecular arrays either in suspension, at air/water interfaces, on planar mono- and bilayer lipid films, on liposomes and on solid supports (e.g. silicon wafers). Detailed studies on composite S-layer/lipid structures have been performed with Langmuir films, freestanding bilayer lipid membranes, solid supported lipid membranes, and liposomes. Lipid molecules in planar films and liposomes interact via their head groups with defined domains on the S-layer lattice. Electrostatic interactions are the most prevalent forces. The hydrophobic chains of the lipid monolayers are almost unaffected by the attachment of the S-layer and no impact on the hydrophobic thickness of the membranes has been observed. Upon crystallization of a coherent S-layer lattice on planar and vesicular lipid membranes, an increase in molecular order is observed, which is reflected in a decrease of the membrane tension and an enhanced mobility of probe molecules within an S-layer-supported bilayer. Thus, the terminology 'semifluid membrane' has been introduced for describing S-layer-supported lipid membranes. The most important feature of composite S-layer/lipid membranes is an enhanced stability in comparison to unsupported membranes.     

Some studies on lipid peroxidation in monomolecular and bimolecular lipid films

Summary Hydrogen peroxide generated from dissolved oxygen through the alloxandialuric acid cycle affected both the permeability and the stability of lipid bilayer membranes. The permeability of the artificial membranes varied directly with the hydrogen peroxide concentration. Membrane stability varied inversely with the hydrogen peroxide concentration. Bilayers formed from solutions containing both phospholipid and the antioxidant vitamin E were less permeable and more stable in the presence of hydrogen peroxide than bilayers generated from solutions containing phospholipid alone. Peroxidation of phospholipid monolayers caused first an expansion of the films presumably through the introduction of peroxide groups. Further oxidation of phospholipid monolayers led to contraction of the films presumably through the formation of water-soluble products. The results of the monolayer studies and a consideration of the possible kinetics for the peroxidation reaction sequence have been used to explain the changes in the permeability and the stability of lipid bilayer membranes. Our data suggest that oxidation of lipid in biological membranes may first increase membrane permeability and then decrease membrane stability.     

Molecular dynamics simulation of a phosphatidylglycerol membrane

Although molecular dynamics simulations are an important tool for studying membrane systems, relatively few simulations have used anionic lipids. This paper reports the first simulation of a pure phosphatidylglycerol (PG) bilayer. The properties of this equilibrated palmitoyloleoylphosphatidylglycerol membrane agree with experimental observations of PG membranes and with previous simulations of monolayers and mixed bilayers containing PG lipids. These simulations also provide interesting insights into hydrogen bonding interactions in PG membranes. This equilibrated membrane will be a useful starting point for simulations of membrane proteins interacting with PG lipids.     

Tarantula toxins interact with voltage sensors within lipid membranes

Voltage-activated ion channels are essential for electrical signaling, yet the mechanism of voltage sensing remains under intense investigation. The voltage-sensor paddle is a crucial structural motif in voltage-activated potassium (K(v)) channels that has been proposed to move at the protein-lipid interface in response to changes in membrane voltage. Here we explore whether tarantula toxins like hanatoxin and SGTx1 inhibit K(v) channels by interacting with paddle motifs within the membrane. We find that these toxins can partition into membranes under physiologically relevant conditions, but that the toxin-membrane interaction is not sufficient to inhibit K(v) channels. From mutagenesis studies we identify regions of the toxin involved in binding to the paddle motif, and those important for interacting with membranes. Modification of membranes with sphingomyelinase D dramatically alters the stability of the toxin-channel complex, suggesting that tarantula toxins interact with paddle motifs within the membrane and that they are sensitive detectors of lipid-channel interactions.     

Multiple binding sites for local anesthetics on reconstituted acetylcholine receptor membranes

Using electron spin resonance spectroscopy and a spin-labeled analog of a tertiary amine local anesthetic, we have identified several populations of the local anesthetic within reconstituted lipid membranes containing purified acetylcholine receptors. These populations represent the local anesthetic interacting with membrane lipid and with the acetylcholine receptor. The data also suggest the existence of at least two classes of binding sites for the local anesthetic on the acetylcholine receptor.     

Exocytosis and membrane recycling

Exocytosis implies the fusion of the membrane of secretion granules with, and the insertion into, the plasmalemma. In non-growing systems such an insertion is temporary in that the inserted membrane is eventually removed. Turnover results indicate that the removed membrane is not destroyed but recycled within the cell and reused. In some systems exocytosis occurs over the entire plasmalemma, while in others it is restricted to discrete regions, characterized by peculiar morphology and composition. Thus the fusion of the two membranes is probably preceded by a recognition step. Structural specializations were detected in interacting granule and plasma membranes by freeze-fracture and surface labelling techniques: arrays of intramembrane particles in protozoans and nerve terminals; clearing of particles and surface antigens in other systems. Direct evidence, obtained in some secretory systems, indicates that after exocytosis the granules and plasma membranes do not intermix, but remain segregated. The subsequent recapture of membrane patches of the granule type (in many systems by means of coated pits and vesicles) could then account for the striking specificity of the recycling process, documented by both composition and structural studies. In different systems the recycling of granule membranes is carried out at greatly different rates. Recent results in the parotid gland and neuromuscular junction indicate that this process is Ca2+-dependent.     

Membrane stability

Rupture and buckling of artificial and biological membranes is an important part of many biological processes. In this review, we present some of the main experimental facts and their analysis. Recent theoretical work, in particular thin film models and nucleation mechanisms of membrane instability, are discussed in detail. Possible applications to membrane adhesion and fusion are pointed out. Attempts are made to explain biological phenomena and experimental results for biological membranes based on a rigorous physicochemical approach developed previously for thin films in colloid systems.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm^?1 and an amide II band at 1548 cm^?1. The outer membrane had a broad amide I band at 1631–1657 cm^?1 and an amid II band at 1548 cm^?1 with a shoulder at 1520–1530 cm^?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm^?1, whereas the band at 1631 cm^?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.     

Effects of phosphatidylethanolamine glycation on lipid-protein interactions and membrane protein thermal stability

Non-enzymatic glycation of biomolecules has been implicated in the pathophysiology of aging and diabetes. Among the potential targets for glycation are biological membranes, characterized by a complex organization of lipids and proteins interacting and forming domains of different size and stability. In the present study, we analyse the effects of glycation on the interactions between membrane proteins and lipids. The phospholipid affinity for the transmembrane surface of the PMCA (plasma-membrane Ca(2+)-ATPase) was determined after incubating the protein or the phospholipids with glucose. Results show that the affinity between PMCA and the surrounding phospholipids decreases significantly after phosphospholipid glycation, but remains unmodified after glycation of the protein. Furthermore, phosphatidylethanolamine glycation decreases by approximately 30% the stability of PMCA against thermal denaturation, suggesting that glycated aminophospholipids induce a structural rearrangement in the protein that makes it more sensitive to thermal unfolding. We also verified that lipid glycation decreases the affinity of lipids for two other membrane proteins, suggesting that this effect might be common to membrane proteins. Extending these results to the in vivo situation, we can hypothesize that, under hyperglycaemic conditions, glycation of membrane lipids may cause a significant change in the structure and stability of membrane proteins, which may affect the normal functioning of membranes and therefore of cells.     

Composite S-layer lipid structures

Designing and utilization of biomimetic membrane systems generated by bottom-up processes is a rapidly growing scientific and engineering field. Elucidation of the supramolecular construction principle of archaeal cell envelopes composed of S-layer stabilized lipid membranes led to new strategies for generating highly stable functional lipid membranes at meso- and macroscopic scale. In this review, we provide a state of the art survey how S-layer proteins, lipids, and polysaccharides may be used as basic building blocks for the assembly of S-layer supported lipid membranes. These biomimetic membrane systems are distinguished by a nanopatterned fluidity, enhanced stability and longevity and thus, provide a dedicated reconstitution matrix for membrane-active peptides and transmembrane proteins. Exciting areas for application of composite S-layer membrane systems concern sensor systems involving specific membrane functions.     

Do proteins facilitate the formation of cholesterol-rich domains?

Both biological and model membranes can exhibit the formation of domains. A brief review of some of the diverse methodologies used to identify the presence of domains in membranes is given. Some of these domains are enriched in cholesterol. The segregation of lipids into cholesterol-rich domains can occur in both pure lipid systems as well as membranes containing peptides and proteins. Peptides and proteins can promote the formation of cholesterol-rich domains not only by preferentially interacting with cholesterol and being sequestered into these regions of the membrane, but also indirectly as a consequence of being excluded from cholesterol-rich domains. The redistribution of components is dictated by the thermodynamics of the system. The formation of domains in a biological membrane is a consequence of all of the intermolecular interactions including those among lipid molecules as well as between lipids and proteins.     

Spin-label ESR studies of lipid-protein interactions in thylakoid membranes

Lipid-protein interactions in thylakoid membranes, and in the subthylakoid membrane fractions containing either photosystem 1 or photosystem 2, have been studied by using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyldiacylglycerol, phosphatidylglycerol, and phosphatidylcholine. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted membrane lipids interacting directly with the integral membrane proteins. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with the membrane proteins and to determine the selectivity between the different lipid classes for the lipid-protein interaction. The fractions of motionally restricted lipid in the thylakoid membrane are 0.36, 0.39, and 0.53, for the spin-labeled monogalactosyldiacylglycerol, phosphatidylcholine, the phosphatidylglycerol, respectively. Spin-labeled monogalactosyldiacylglycerol exhibits very little preferential interaction over phosphatidylchline, which suggests that part of the role of monogalactosyldiacylglycerol in thylakoid membranes is structural, as is the case for phosphatidylcholine in mammalian membranes. Spin-labeled phosphatidylglycerol shows a preferential interaction over the corresponding monogalactosyldiacylglycerol and phosphatidylcholine analogues, in contrast to the common behavior of this lipid in mammalian systems. This pattern of lipid selectivity is preserved in both the photosystem 1 and photosystem 2 enriched subthylakoid membrane fractions.     

The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers

The association of bacterial lipopolysaccharide with artificial membranes was studied in an attempt to understand the mechanism of binding of lipopolysaccharide to cell surfaces and to look for an effect on membrane stability. The membrane models used were phospholipid bilayers and monolayers. As measured by survival time, lipopolysaccharide was found to decrease the stability of bilayers at a concentration of 300 μg/ml. When assayed by dielectric breakdown, an effect of lipopolysaccharide was noticeable at concentrations of 50 μg/ml. In studies involving the penetration of monomolecular films of various phospholipids, native and alkali-treated lipopolysaccharide both caused increases in surface pressure, and therefore penetrated the films. However, alkali-treated lipopolysaccharide was at least ten times more efficient than the native product in penetration. Alkali-treated lipopolysaccharide had a greater degree of surface activity than native lipopolysaccharide, since alkali-treated lipopolysaccharide formed monomolecular films by itself, whereas native lipopolysaccharide did not. The changes in the surface pressure and surface potential of phospholipid films produced by lipopolysaccharide in the subsolution suggested that the interaction of lipopolysaccharide with phospholipid monolayers was by a combination of penetration and adsorption to the undersurface.     

Layer-by-layer Synthesis and Transfer of Freestanding Conjugated Microporous Polymer Nanomembranes

CMP as large surface area materials have attracted growing interest recently, due to their high variability in the incorporation of functional groups in combination with their outstanding thermal and chemical stability, and low densities. However, their insoluble nature causes problems in their processing since usually applied techniques such as spin coating are not available. Especially for membrane applications, where the processing of CMP as thin films is desirable, the processing problems have hindered their commercial application.Here we describe the interfacial synthesis of CMP thin films on functionalized substrates via molecular layer-by-layer (l-b-l) synthesis. This process allows the preparation of films with desired thickness and composition and even desired composition gradients.The use of sacrificial supports allows the preparation of freestanding membranes by dissolution of the support after the synthesis. To handle such ultra-thin freestanding membranes the protection with sacrificial coatings showed great promise, to avoid rupture of the nanomembranes. To transfer the nanomembranes to the desired substrate, the coated membranes are upfloated at the air-liquid interface and then transferred via dip coating.     

The study on the interaction between phytosterols and phospholipids in model membranes

Sterols are one of the major components of cellular membranes. Although in mammalian membranes cholesterol is a predominant sterol, in the human organism plant sterols (phytosterols) can also be found. Phytosterols, especially if present in concentrations higher than normal (phytosterolemia), may strongly affect membrane properties. In this work, we studied phytosterol-phospholipid interactions in mixed Langmuir monolayers serving as model membranes. Investigated were two phytosterols, beta-sitosterol and stigmasterol and a variety of phospholipids, both phosphatidylethanolamines and phosphatidylcholines. The phospholipids had different polar heads, different length and saturation of their hydrocarbon chains. The interactions between molecules in mixed sterol/phospholipid films were characterized with the mean area per molecule (A(12)) and the excess free energy of mixing (DeltaG(Exc)). The effect of the sterols on the molecular organization of the phospholipid monolayers was analyzed based on the compression modulus values. It was found that the incorporation of the phytosterols into the phospholipid monolayers increased their condensation. The plant sterols revealed higher affinity towards phosphatidylcholines as compared to phosphatidylethanolamines. The phytosterols interacted more strongly with phospholipids possessing longer and saturated chains. Moreover, both the length and the saturation of the phosphatidylcholines influenced the stoichiometry of the most stable complexes. Our results, compared with those presented previously for cholesterol/phospholipid monolayers, allowed us to draw a conclusion that the structure of sterol (cholesterol, beta-sitosterol, stigmasterol) does not affect the stoichiometry of the most stable complexes formed with particular phospholipids, but influences their stability. Namely, the strongest interactions were found for cholesterol/phospholipids mixtures, while the weakest for mixed systems containing stigmasterol.     

Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.     

SNARE protein analog‐mediated membrane fusion

Fusion of lipid membranes to form a single bilayer is an essential process for life and provides important biological functions including neurotransmitter release. Membrane fusion proteins facilitate approximation of interacting membranes to overcome the energy barrier. In case of synaptic transmission, proteins involved are known as soluble N‐ethylmaleimide‐sensitive‐factor attachment receptor (SNARE) proteins. The SNAREs from synaptic vesicles interact with the SNAREs from the target membrane to form a coiled‐coil bundle of four helices, thus pulling the membranes tightly together and initiating fusion. However, it remains unclear how these proteins function at molecular level. Natural systems are often too complex to obtain unambiguous results. Simple model systems mimicking natural proteins in synthetic lipid bilayers are powerful tools for obtaining insights into this essential biological process. An important advantage of such systems is their well‐defined composition, which can be systematically varied in order to fully understand events at molecular level. In this review, selected model systems are presented based upon specific interactions between recognition units embedded in separate lipid bilayers mimicking native SNARE protein‐mediated membrane fusion. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of two oxysterols, 7-ketocholesterol and 25-hydroxycholesterol,with phosphatidylcholine and sphingomyelin in model membranes

Oxidized analogs of cholesterol (oxysterols) are produced through both enzymatic and non-enzymatic pathways and have been shown to perturb membrane properties in vitro and in vivo. In the present study, the membrane behavior of two naturally occurring oxysterols, 25-hydroxycholesterol and 7-ketocholesterol, was examined in two model systems. The presence of an additional oxygen moiety was found to alter membrane properties compared to native cholesterol and to each other in lipid monolayers, composed of either pure sterol or sterol–glycerophospholipid and sterol–sphingomyelin binary films, as well as in mixed multilamellar vesicles. The ability of oxysterols to condense phosphatidylcholine and sphingomyelin films, their capacity to cause changes in in-plane elasticity moduli, and their propensity to form detergent-resistant membrane domains were all found to be dependant on the location of the oxygen functionality in the oxysterol, the chemical nature of the phospholipid in the model systems, and the oxysterol/phospholipid ratio in the membrane. The findings described in this study with respect to their biophysical/biophysiological implications provide additional insight into the activity of cytotoxic oxysterols in model membranes.     

Infrared techniques for quantifying protein structural stability

Biopharmaceutical and biotechnology companies and regulatory agencies require novel methods to determine the structural stabilities of proteins and the integrity of protein-protein, protein-ligand, and protein-membrane interactions that can be applied to a variety of sample states and environments. Infrared spectroscopy is a favorable method for a number of reasons: it is adequately sensitive to minimal sample amounts and is not limited by the molecular weight of the sample; yields spectra that are simple to evaluate; does not require protein modifications, a special supporting matrix, or internal standard; and is applicable to soluble and membrane proteins. In this paper, we investigate the application of infrared spectroscopy to the quantification of protein structural stability by measuring the extent of amide hydrogen/deuterium exchange in buffers containing D₂O for proteins in solution and interacting with ligands and lipid membranes. We report the thermodynamic stability of several protein preparations, including chick egg-white lysozyme, trypsin bound by benzamidine inhibitors, and cytochrome c interacting with lipid membranes of varying net-negative surface charge density. The results demonstrate that infrared spectroscopy can be used to compare protein stability as determined by amide hydrogen/deuterium exchange for a variety of cases.     

Interaction of Trastuzumab with biomembrane models at air-water interfaces mimicking cancer cell surfaces

Trastuzumab (Tmab) is a monoclonal antibody administered as targeted therapy for HER2-positive breast cancer whose molecular interactions at the HER2 receptor microenvironment are not completely clarified yet. This paper describes the influence of Tmab in the molecular organization of films of biological-relevant molecules at the air water interface. For that, we spread components of tumorigenic and non-tumorigenic cells directly on the air-water interface. The physicochemical properties of the films were investigated with surface pressure-area isotherms and Brewster angle microscopy, and distinction between the cellular lines with higher or lower amount of HER2 could be detected based on the physicochemical properties of the interfacial films. The systems organized at the air-water interface were transferred to solid supports as Langmuir-Blodgett films and the nano-scale morphology investigated with atomic force microscopy. The overall results related to Tmab interacting with the films lead to the conclusion that Tmab tends to condense rich-HER2 films, causing irregular dimerization of the receptor protein, changing the membrane topography of the films, with formation of phases with different levels of reflectivity and aggregation morphology, and finally revealing that the interaction of the antibody with proteo-lipidic biointerfaces is modulated by the film composition. We believe that novel perspectives concerning the molecular interactions in the plasma membrane microenvironment through Langmuir monolayers can be obtained from this work in order to enhance the Tmab-based cancer therapy.