RESUMEN
Rapid and precise detection of significant data streams within a network is crucial for efficient traffic management. This study leverages the TabNet deep learning architecture to identify large-scale flows, known as elephant flows, by analyzing the information in the 5-tuple fields of the initial packet header. The results demonstrate that employing a TabNet model can accurately identify elephant flows right at the start of the flow and makes it possible to reduce the number of flow table entries by up to 20 times while still effectively managing 80% of the network traffic through individual flow entries. The model was trained and tested on a comprehensive dataset from a campus network, demonstrating its robustness and potential applicability to varied network environments.
RESUMEN
Reconstitution of a transmembrane protein in model lipid systems allows studying its structure and dynamics in isolation from the complexity of the natural environment. This approach also provides a well-defined environment for studying the interactions of proteins with lipids. In this work, we describe the FRET-GP method, which utilizes Förster resonance energy transfer (FRET) to specifically probe the nanoenvironment of a transmembrane domain. The tryptophan residues flanking this domain act as efficient FRET donors, while Laurdan acts as acceptor. The fluorescence of this solvatochromic probe is quantified using generalized polarization (GP) to report on lipid mobility in the vicinity of the transmembrane domain. We applied FRET-GP to study the transmembrane peptide WALP incorporated in liposomes. We found that the direct excitation of Laurdan to its second singlet state strongly contributes to GP values measured in FRET conditions. Removal of this parasitic contribution was essential for proper determination of GPFRET - the local analogue of classical GP parameter. The presence of WALP significantly increased both parameters but the local effects were considerably stronger (GPFRET â« GP). We conclude that WALP restricts lipid movement in its vicinity, inducing lateral inhomogeneity in membrane fluidity. WALP was also found to influence lipid phase transition. Our findings demonstrated that FRET-GP simultaneously provides local and global results, thereby enhancing the depth of information obtained from the measurement. We highlight the simplicity and sensitivity of the method, but also discuss its potential and limitations in studying protein-lipid interactions.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Lauratos , Liposomas/química , Péptidos , Colorantes FluorescentesRESUMEN
Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.
Asunto(s)
Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Fusión de Membrana/fisiología , Arginina/metabolismo , Arginina/fisiología , Transporte Biológico , Membrana Celular/metabolismo , Cinética , Membrana Dobles de Lípidos/química , Fusión de Membrana/efectos de los fármacos , Membranas/metabolismo , Simulación de Dinámica Molecular , Péptidos/química , Péptidos/fisiología , Seudópodos/metabolismo , Seudópodos/fisiologíaRESUMEN
Cholesterol renders mammalian cell membranes more compact by reducing the amount of voids in the membrane structure. Because of this, cholesterol is known to regulate the ability of cell membranes to prevent the permeation of water and water-soluble molecules through the membranes. Meanwhile, it is also known that even seemingly tiny modifications in the chemical structure of cholesterol can lead to notable changes in membrane properties. The question is, how significantly do these small changes in cholesterol structure affect the permeability barrier function of cell membranes? In this work, we applied fluorescence methods as well as atomistic molecular dynamics simulations to characterize changes in lipid membrane permeability induced by cholesterol oxidation. The studied 7ß-hydroxycholesterol (7ß-OH-chol) and 27-hydroxycholesterol (27-OH-chol) represent two distinct groups of oxysterols, namely, ring- and tail-oxidized cholesterols, respectively. Our previous research showed that the oxidation of the cholesterol tail has only a marginal effect on the structure of a lipid bilayer; however, oxidation was found to disturb membrane dynamics by introducing a mechanism that allows sterol molecules to move rapidly back and forth across the membrane-bobbing. Herein, we show that bobbing of 27-OH-chol accelerates fluorescence quenching of NBD-lipid probes in the inner leaflet of liposomes by dithionite added to the liposomal suspension. Systematic experiments using fluorescence quenching spectroscopy and microscopy led to the conclusion that the presence of 27-OH-chol increases membrane permeability to the dithionite anion. Atomistic molecular dynamics simulations demonstrated that 27-OH-chol also facilitates water transport across the membrane. The results support the view that oxysterol bobbing gives rise to successive perturbations to the hydrophobic core of the membrane, and these perturbations promote the permeation of water and small water-soluble molecules through a lipid bilayer. The observed impairment of permeability can have important consequences for eukaryotic organisms. The effects described for 27-OH-chol were not observed for 7ß-OH-chol which represents ring-oxidized sterols.
RESUMEN
Cytosine 2'-deoxyribonucleoside dCTBdp and its triphosphate (dCTBdpTP) bearing tetramethylated thiophene-bodipy fluorophore attached at position 5 were designed and synthesized. The green fluorescent nucleoside dCTBdp showed a perfect dependence of fluorescence lifetime on the viscosity. The modified triphosphate dCTBdpTP was substrate to several DNA polymerases and was used for in vitro enzymatic synthesis of labeled oligonucleotides (ONs) or DNA by primer extension. The labeled single-stranded ONs showed a significant decrease in mean fluorescence lifetime when hybridized to the complementary strand of DNA or RNA and were also sensitive to mismatches. The labeled dsDNA sensed protein binding (p53), which resulted in the increase of its fluorescence lifetime. The triphosphate dCTBdpTP was transported to live cells where its interactions could be detected by FLIM but it did not show incorporation to genomic DNA in cellulo.
Asunto(s)
Compuestos de Boro/química , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Hibridación de Ácido Nucleico , Nucleótidos/química , Sondas de Oligonucleótidos/metabolismo , Tiofenos/química , Secuencia de Bases , Cationes , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Lípidos/química , Nucleótidos/síntesis química , Unión Proteica , Solventes/química , Espectrometría de Fluorescencia , Temperatura , ViscosidadRESUMEN
Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.
Asunto(s)
Lípidos de la Membrana/química , Microdominios de Membrana/química , Nanoestructuras/química , FluorescenciaRESUMEN
Regulation of cell metabolism, membrane fusion, association of proteins with cellular membranes, and cellular signaling altogether would not be possible without Ca2+ ions. The distribution of calcium within the cell is uneven with the negatively charged inner leaflet of the plasma membrane being one of the primary targets of its accumulation. Therefore, we decided to map the influence of Ca2+ on the properties of lipid bilayers closely resembling natural lipid membranes. We combined fluorescence spectroscopy (analysis of time-resolved emission spectra of Laurdan probe and derived parameters: integrated relaxation time related to local lipid mobility, and total emission shift reflecting membrane polarity and hydration) with molecular dynamics simulations to determine the effect of the increasing CaCl2 concentration on model lipid membranes containing POPC, POPS, and cholesterol. On top of that, the impact of calcium on the plasma membranes isolated from HEK293 cells was investigated using the steady-state fluorescence of Laurdan. We found that calcium increases rigidity of all the model lipid membranes used, elevates their thickness, increases lipid packing and ordering, and impedes the local lipid mobility. All these effects were to a great extent similar to those elicited by cholesterol. However, the changes of the membrane properties induced by calcium and cholesterol seem largely independent from each other. At sufficiently high concentrations of calcium or cholesterol, the steric effects hindered a further alteration of membrane organization, i.e., the compressibility limit of membrane structures was reached. We found no indication for mutual interaction between Ca2+ and cholesterol, nor competition of Ca2+ ions and hydroxyl groups of cholesterol for binding to phospholipids. Fluorescence measurements indicated that Ca2+ adsorption decreases mobility within the carbonyl region of model bilayers more efficiently than monovalent ions do (Ca2+ â« Li+ > Na+ > K+ > Cs+). The effects of calcium ions were to a great extent mitigated in the plasma membranes isolated from HEK293 cells when compared to the model lipid membranes. Noticeably, the plasma membranes showed remarkably higher resistance toward rigidification induced by calcium ions even when compared with the model membranes containing cholesterol.
Asunto(s)
Calcio/química , Membrana Celular/química , Colesterol/química , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Células HEK293 , Humanos , Simulación de Dinámica Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
Cyanidin and its O-glycosides have many important physiological functions in plants and beneficial effects on human health. Their biological activity is not entirely clear and depends on the structure of the molecule, in particular, on the number and type of sugar substituents. Therefore, in this study the detailed structure-activity relationship (SARs) of the anthocyanins/anthocyanidins in relation to their interactions with lipid bilayer was determined. On the basis of their antioxidant activity and the changes induced by them in size and Zeta potential of lipid vesicles, and mobility and order of lipid acyl chains, the impact of the number and type of sugar substituents on the biological activity of the compounds was evaluated. The obtained results have shown, that 3-O-glycosylation changes the interaction of cyanidin with lipid bilayer entirely. The 3-O-glycosides containing a monosaccharide induces greater changes in physical properties of the lipid membrane than those containing disaccharides. The presence of additional sugar significantly reduces glycoside interaction with model lipid membrane. Furthermore, O-glycosylation alters the ability of cyanidin to scavenge free radicals. This alteration depends on the type of free radicals and the sensitivity of the method used for their determination.
Asunto(s)
Antocianinas/metabolismo , Fosfatidilcolinas/metabolismo , Glicosilación , Peróxido de Hidrógeno/metabolismo , Peroxidación de Lípido , Relación Estructura-ActividadRESUMEN
Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquidâ»crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquidâ»crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.
Asunto(s)
1,2-Dipalmitoilfosfatidilcolina/química , 2-Naftilamina/análogos & derivados , Lauratos/química , Membrana Dobles de Lípidos/química , Modelos Químicos , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , 2-Naftilamina/química , Fluorescencia , Teoría CuánticaRESUMEN
Procyanidins, contained in many products abundant in human diet, exhibit high biological activity. However, this activity has not been fully explained at cellular and molecular levels. In this study, we determine the mechanism of interaction of procyanidin B3 with model lipid membrane. This mechanism was established on the basis of changes induced by B3 in the physical properties of lipid bilayer. The changes were investigated using steady state and time-resolved fluorescence, DSC, and FTIR. We show that procyanidin B3 causes changes in the arrangement of the polar heads of lipids, order of their acyl chains and the main lipid phase transition temperature. Furthermore, its presence in the membrane leads to a reduction in membrane dipole potential. Procyanidin B3 is anchored to membrane via hydrogen bonds formed between its OH groups and the PO2- and CO groups of lipids, causing changes in both hydrophilic and hydrophobic regions of the membrane.
Asunto(s)
2-Naftilamina/análogos & derivados , Biflavonoides/química , Catequina/química , Dihidropiridinas/química , Dimiristoilfosfatidilcolina/química , Lauratos/química , Membrana Dobles de Lípidos/química , Proantocianidinas/química , 2-Naftilamina/química , Rastreo Diferencial de Calorimetría , Enlace de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Transición de Fase , Espectrometría de Fluorescencia , Espectroscopía Infrarroja por Transformada de Fourier , Temperatura , TermodinámicaRESUMEN
Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.
Asunto(s)
Ésteres del Colesterol/química , Colesterol/química , Liposomas/química , Protones , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Dihidropiridinas/química , Lauratos/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Fosfatidilcolinas/química , Agua/químicaRESUMEN
Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso-substituted BODIPY fluorescent molecular rotor (dC(bdp)) to sense changes in DNA microenvironment both inâ vitro and in living cells. DNA incorporating dC(bdp) can respond to interactions with DNA-binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5-2.2â ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA-associated processes, cellular structures, and also DNA-based nanomaterials.
Asunto(s)
Compuestos de Boro/química , ADN/química , Fluorescencia , Colorantes Fluorescentes/química , Microscopía Confocal/métodos , Nucleótidos/química , Rotación , ADN/síntesis química , Células HeLa , Humanos , Nanoestructuras/químicaRESUMEN
Recently, we reported the use of the Nicking Enzyme Amplification Reaction (NEAR) for the enzymatic synthesis of short oligonucleotides (ONs) containing 5-substituted pyrimidine or 7-substituted 7-deazaadenine nucleotides. Since no oligonucleotide products were visible on agarose gels stained by an intercalating dye (GelRed), we assumed that the method did not work for 7-substituted 7-deazaguanine deoxyribonucleoside triphosphates. We revisited the work and found that the NEAR method works for 7-deazaguanine nucleotides as well but that the resulting modified ONs quench the fluorescence of DNA intercalators, rendering them invisible on gel electrophoresis stained by them. Here, we report on the modified methodology for the NEAR synthesis and analysis of G-modified ONs and on quantification of the fluorescence quenching.
Asunto(s)
Colorantes Fluorescentes/química , Guanina/análogos & derivados , Oligonucleótidos/química , Secuencia de Bases , Fluorescencia , Guanina/síntesis química , Guanina/química , Oligonucleótidos/síntesis química , Espectrometría de FluorescenciaRESUMEN
Fluorescence methods are versatile tools for obtaining dynamic and topological information about biomembranes because the molecular interactions taking place in lipid membranes frequently occur on the same timescale as fluorescence emission. The fluorescence intensity decay, in particular, is a powerful reporter of the molecular environment of a fluorophore. The fluorescence lifetime can be sensitive to the local polarity, hydration, viscosity, and/or presence of fluorescence quenchers/energy acceptors within several nanometers of the vicinity of a fluorophore. Illustrative examples of how time-resolved fluorescence measurements can provide more valuable and detailed information about a system than the time-integrated (steady-state) approach will be presented in this review: 1), determination of membrane polarity and mobility using time-dependent spectral shifts; 2), identification of submicroscopic domains by fluorescence lifetime imaging microscopy; 3), elucidation of membrane leakage mechanisms from dye self-quenching assays; and 4), evaluation of nanodomain sizes by time-resolved Förster resonance energy transfer measurements.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Cinética , Microscopía Fluorescente/métodosRESUMEN
Positioning of peptides with respect to membranes is an important parameter for biological and biophysical studies using model systems. Our experiments using five different membrane peptides suggest that the time-dependent fluorescence shift (TDFS) of Laurdan can help when distinguishing between peripheral and integral membrane binding and can be a useful, novel tool for studying the impact of transmembrane peptides (TMP) on membrane organization under near-physiological conditions. This article focuses on LAH4, a model α-helical peptide with high antimicrobial and nucleic acid transfection efficiencies. The predominantly helical peptide has been shown to orient in supported model membranes parallel to the membrane surface at acidic and, in a transmembrane manner, at basic pH. Here we investigate its interaction with fully hydrated large unilamellar vesicles (LUVs) by TDFS and fluorescence correlation spectroscopy (FCS). TDFS shows that at acidic pH LAH4 does not influence the glycerol region while at basic pH it makes acyl groups at the glycerol level of the membrane less mobile. TDFS experiments with antimicrobial peptides alamethicin and magainin 2, which are known to assume transmembrane and peripheral orientations, respectively, prove that changes in acyl group mobility at the glycerol level correlate with the orientation of membrane-associated peptide molecules. Analogous experiments with the TMPs LW21 and LAT show similar effects on the mobility of those acyl groups as alamethicin and LAH4 at basic pH. FCS, on the same neutral lipid bilayer vesicles, shows that the peripheral binding mode of LAH4 is more efficient in bilayer permeation than the transmembrane mode. In both cases, the addition of LAH4 does not lead to vesicle disintegration. The influence of negatively charged lipids on the bilayer permeation is also addressed.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/química , Péptidos/química , Alameticina/química , Diseño de Fármacos , Glicerol/química , Humanos , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Lípidos/química , Magaininas/química , Espectroscopía de Resonancia Magnética , Conformación Proteica , Espectrometría de Fluorescencia , Factores de Tiempo , Liposomas Unilamelares/químicaRESUMEN
We claim that (1) cholesterol protects bilayers from disruption caused by lipid oxidation by sequestering conical shaped oxidized lipid species such as 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PZPC) away from phospholipid, because cholesterol and the oxidized lipid have complementary shapes and (2) mixtures of cholesterol and oxidized lipids can self-assemble into bilayers much like lysolipidcholesterol mixtures. The evidence for bilayer protection comes from molecular dynamics (MD) simulations and dynamic light scattering (DLS) measurements. Unimodal size distributions of extruded vesicles (LUVETs) made up of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and PZPC containing high amounts of PZPC are only obtained when cholesterol is present in high concentrations. In simulations, bilayers containing high amounts of PZPC become porous, unless cholesterol is also present. The protective effect of cholesterol on oxidized lipids has been observed previously using electron paramagnetic resonance (EPR) and electron microscopy imaging of vesicles. The evidence for the pairing of cholesterol and PZPC comes mainly from correlated 2-D density and thickness plots from simulations, which show that these two molecules co-localize in bilayers. Further evidence that the two molecules can cohabitate comes from self-assembly simulations, where we show that cholesterol-oxidized lipid mixtures can form lamellar phases at specific concentrations, reminiscent of lysolipidcholesterol mixtures. The additivity of the packing parameters of cholesterol and PZPC explains their cohabitation in a planar bilayer. Oxidized lipids are ubiquitously present in significant amounts in high- and low-density lipoprotein (HDL and LDL) particles, diseased tissues, and in model phospholipid mixtures containing polyunsaturated lipids. Therefore, our hypothesis has important consequences for cellular cholesterol trafficking; diseases related to oxidized lipids, and to biophysical studies of phase behaviour of cholesterol-containing phospholipid mixtures.
Asunto(s)
Colesterol/química , Membrana Dobles de Lípidos/química , Fosforilcolina/análogos & derivados , Exosomas/química , Oxidación-Reducción , Fosfatidilcolinas/química , Fosforilcolina/químicaRESUMEN
The environment-sensitive fluorescent probes provide excellent tools for studying membranes in their native state. We have modified the BODIPY-based fluorescent molecular rotor by increasing the number of alkyl moieties from one to two or three to achieve a more defined and deeper positioning of the probe in membranes. Detailed characterisation of fluorescence properties and localisation/orientation of probes was performed using a variety of fluorescence techniques and model membranes composed of different lipids. As expected, additional alkyls attached to the fluorophore moiety led to a deeper and more defined localisation of the probe in the lipid bilayer. The results strongly indicate that fluorescence properties of such probes are influenced not only by lipid packing but also by the orientation of the probe in membranes. The orientation of rotors studied herein was significantly altered by changes in the lipid composition of membranes. Our observations demonstrate the limits of BODIPY-based molecular rotors as environmental sensors in cellular membranes with complex lipid composition. The results presented herein also underline the importance of the detailed characterisation of fluorescent membrane dyes and provide a guide for future testing.
Asunto(s)
Compuestos de Boro/química , Colorantes Fluorescentes/química , Membrana Dobles de Lípidos/química , Colorantes Fluorescentes/síntesis química , Membrana Dobles de Lípidos/síntesis química , Estructura MolecularRESUMEN
Time-dependent fluorescence shift (TDFS) of Laurdan embedded in phospholipid bilayers reports on hydration and mobility of the phospholipid acylgroups. Exchange of H2O with D2O prolongs the lifetime of lipid-water and lipid-water-lipid interactions, which is reflected in a significantly slower TDFS kinetics. Combining TDFS measurements in H2O and D2O hydrated bilayers with atomistic molecular dynamics (MD) simulations provides a unique tool for characterization of the hydrogen bonding at the acylgroup level of lipid bilayers. In this work, we use this approach to study the influence of fluoride anions on the properties of cationic bilayers composed of trimethylammonium-propane (DOTAP). The results obtained for DOTAP are confronted with those for neutral phosphatidylcholine (DOPC) bilayers. Both in DOTAP and DOPC H2O/D2O exchange prolongs hydrogen-bonding lifetime and does not disturb bilayer structure. These results are confirmed by MD simulations. TDFS experiments show, however, that for DOTAP this effect is cancelled in the presence of fluoride ions. We interpret these results as evidence that strongly hydrated fluoride is able to steal water molecules that bridge lipid carbonyls. Consequently, when attracted to DOTAP bilayer, fluoride disrupts the local hydrogen-bonding network, and the differences in TDFS kinetics between H2O and D2O hydrated bilayers are no longer observed. A distinct behavior of fluoride is also evidenced by MD simulations, which show different lipid-ion binding for Cl(-) and F(-).
Asunto(s)
Ácidos Grasos Monoinsaturados/química , Fluoruros/química , Membrana Dobles de Lípidos/química , Simulación de Dinámica Molecular , Compuestos de Amonio Cuaternario/química , Agua/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/análisis , Colorantes Fluorescentes/análisis , Enlace de Hidrógeno , Lauratos/análisis , Fosfatidilcolinas/químicaRESUMEN
Transmembrane (TM) proteins interact closely with the surrounding membrane lipids. Lipids in the vicinity of TM proteins were reported to have hindered mobility, which has been associated with lipids being caught up in the rough surface of the TM domains. These reports, however, neglect one important factor that largely influences the membrane behavior - electrostatics of the TM peptides that are usually positively charged at their cytosolic end. Here, we study on the example of a neutral and a positively charged WALP peptide, how the charge of a TM peptide influences the membrane. We investigate both its dynamics and mechanics by: (i) time dependent fluorescent shift in combination with classical and FRET generalized polarization to evaluate the mobility of lipids at short and long-range distance from the peptide, (ii) atomic force microscopy to observe the mechanical stability of the peptide-containing membranes, and (iii) molecular dynamics simulations to analyze the peptide-lipid interactions. We show that both TM peptides lower lipid mobility in their closest surroundings. The peptides cause lateral heterogeneity in lipid mobility, which in turn prevents free lipid rearrangement and lowers the membrane ability to seal ruptures after mechanical indentations. Introduction of a positive charge to the peptide largely enhances these effects, affecting the whole membrane. We thus highlight that unspecific peptide-lipid interactions, especially the electrostatics, should not be overlooked as they have a great impact on the mechanics and dynamics of the whole membrane.
Asunto(s)
Membrana Dobles de Lípidos , Péptidos , Membrana Dobles de Lípidos/química , Péptidos/química , Lípidos de la Membrana/química , Proteínas de la Membrana/química , Simulación de Dinámica MolecularRESUMEN
Effects of alkali metal chlorides on the properties of mixed negatively charged lipid bilayers are experimentally measured and numerically simulated. Addition of 20mol% of negatively charged phosphatidylserine to zwitterionic phosphatidylcholine strengthens adsorption of monovalent cations revealing their specificity, in the following order: Cs(+)Asunto(s)
Cesio/química
, Cloruros/química
, Membrana Dobles de Lípidos/química
, Fosfatidilcolinas/química
, Fosfatidilserinas/química
, Cloruro de Potasio/química
, Cloruro de Sodio/química