Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 16 de 16
Filtrar
1.
Langmuir ; 35(35): 11358-11368, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31393734

RESUMO

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+ ≫ 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.


Assuntos
Cálcio/química , Membrana Celular/química , Colesterol/química , Bicamadas Lipídicas/química , Fosfolipídeos/química , Células HEK293 , Humanos , Simulação de Dinâmica Molecular , Tamanho da Partícula , Propriedades de Superfície
2.
Phys Chem Chem Phys ; 21(4): 1682-1688, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30418442

RESUMO

Nitrobenzoxadiazole (NBD) labeled lipids are popular fluorescent probes of membrane structure and dynamics, and have been widely used in both model systems and living cells. Irrespective of attachment to the lipid head group or hydrocarbon chains, the NBD fluorophore generally adopts a transverse bilayer location near the host lipid carbonyl/glycerol moieties. Still, considerable variability is observed in the measured fluorescence lifetimes, indicating that overall fluorophore location is not the determinant of NBD fluorescence properties. Combining fluorescence experiments and molecular dynamics simulations, we show that for two almost identical NBD probes, significant differences in fluorophore orientation and fluorescence lifetime are observed. Integrating these findings with literature data, we demonstrate a correlation between NBD orientation and fluorescence lifetime. The latter is longer when the NBD nitro group is predominantly oriented towards the bilayer interior, compared to probes for which it points to the water medium.


Assuntos
Bicamadas Lipídicas , Fosforilcolina , Corantes Fluorescentes , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Nitrobenzenos/química , Fosforilcolina/química , Espectrometria de Fluorescência
3.
Biophys J ; 112(10): 2147-2158, 2017 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-28538152

RESUMO

Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using 31P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.


Assuntos
Apoptose/fisiologia , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Proteína X Associada a bcl-2/metabolismo , Varredura Diferencial de Calorimetria , Cardiolipinas/metabolismo , Permeabilidade da Membrana Celular , Escherichia coli , Corantes Fluorescentes , Humanos , Bicamadas Lipídicas/química , Ressonância Magnética Nuclear Biomolecular , Oxirredução , Estresse Oxidativo/fisiologia , Fosfolipídeos/metabolismo , Temperatura , Lipossomas Unilamelares/química
4.
Biochim Biophys Acta ; 1858(6): 1288-97, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26947183

RESUMO

Mitochondria play a crucial role in programmed cell death via the intrinsic apoptotic pathway, which is tightly regulated by the B-cell CLL/lymphoma-2 (Bcl-2) protein family. Intracellular oxidative stress causes the translocation of Bax, a pro-apoptotic family member, to the mitochondrial outer membrane (MOM) where it induces membrane permeabilization. Oxidized phospholipids (OxPls) generated in the MOM during oxidative stress directly affect the onset and progression of mitochondria-mediated apoptosis. Here we use MOM-mimicking lipid vesicles doped with varying concentrations of 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), an OxPl species known to significantly enhance Bax-membrane association, to investigate three key aspects of Bax's action at the MOM: 1) induction of Bax pores in membranes without additional mediator proteins, 2) existence of a threshold OxPl concentration required for Bax-membrane action and 3) mechanism by which PazePC disturbs membrane organization to facilitate Bax penetration. Fluorescence leakage studies revealed that Bax-induced leakage, especially its rate, increased with the vesicles' PazePC content without any detectable threshold neither for OxPl nor Bax. Moreover, the leakage rate correlated with the Bax to lipid ratio and the PazePC content. Solid state NMR studies and calorimetric experiments on the lipid vesicles confirmed that OxPl incorporation disrupted the membrane's organization, enabling Bax to penetrate into the membrane. In addition, 15N cross polarization (CP) and insensitive nuclei enhanced by polarization transfer (INEPT) MAS NMR experiments using uniformly (15)N-labeled Bax revealed dynamically restricted helical segments of Bax embedded in the membrane, while highly flexible protein segments were located outside or at the membrane surface.


Assuntos
Membranas Mitocondriais/metabolismo , Fosforilcolina/análogos & derivados , Proteína X Associada a bcl-2/metabolismo , Varredura Diferencial de Calorimetria , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Humanos , Oxirredução , Permeabilidade , Fosforilcolina/metabolismo , Espectroscopia de Prótons por Ressonância Magnética , Lipossomas Unilamelares
5.
Biochim Biophys Acta ; 1848(2): 444-52, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25450349

RESUMO

Didehydroroflamycoin (DDHR), a recently isolated member of the polyene macrolide family, was shown to have antibacterial and antifungal activity. However, its mechanism of action has not been investigated. Antibiotics from this family are amphiphilic; thus, they have membrane activity, their biological action is localized in the membrane, and the membrane composition and physical properties facilitate the recognition of a particular compound by the target organism. In this work, we use model lipid membranes comprised of giant unilamellar vesicles (GUVs) for a systematic study of the action of DDHR. In parallel, experiments are conducted using filipin III and amphotericin B, other members of the family, and the behavior observed for DDHR is described in the context of that of these two heavily studied compounds. The study shows that DDHR disrupts membranes via two different mechanisms and that the involvement of these mechanisms depends on the presence of cholesterol. The leakage assays performed in GUVs and the conductance measurements using black lipid membranes (BLM) reveal that the pores that develop in the absence of cholesterol are transient and their size is dependent on the DDHR concentration. In contrast, cholesterol promotes the formation of more defined structures that are temporally stable.


Assuntos
Antibacterianos/química , Antifúngicos/química , Filipina/análogos & derivados , Bicamadas Lipídicas/química , Macrolídeos/química , Lipossomas Unilamelares/química , Anfotericina B/química , Colesterol/química , Filipina/química , Cinética
6.
J Chem Phys ; 141(22): 22D516, 2014 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-25494787

RESUMO

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(-).


Assuntos
Ácidos Graxos Monoinsaturados/química , Fluoretos/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Compostos de Amônio Quaternário/química , Água/química , 2-Naftilamina/análogos & derivados , 2-Naftilamina/análise , Corantes Fluorescentes/análise , Ligação de Hidrogênio , Lauratos/análise , Fosfatidilcolinas/química
7.
Artigo em Inglês | MEDLINE | ID: mdl-37271251

RESUMO

Lipid droplets (LD) are important regulators of lipid metabolism and are implicated in several diseases. However, the mechanisms underlying the roles of LD in cell pathophysiology remain elusive. Hence, new approaches that enable better characterization of LD are essential. This study establishes that Laurdan, a widely used fluorescent probe, can be used to label, quantify, and characterize changes in cell LD properties. Using lipid mixtures containing artificial LD we show that Laurdan GP depends on LD composition. Accordingly, enrichment in cholesterol esters (CE) shifts Laurdan GP from ∼0.60 to ∼0.70. Moreover, live-cell confocal microscopy shows that cells present multiple LD populations with distinctive biophysical features. The hydrophobicity and fraction of each LD population are cell type dependent and change differently in response to nutrient imbalance, cell density, and upon inhibition of LD biogenesis. The results show that cellular stress caused by increased cell density and nutrient overload increased the number of LD and their hydrophobicity and contributed to the formation of LD with very high GP values, likely enriched in CE. In contrast, nutrient deprivation was accompanied by decreased LD hydrophobicity and alterations in cell plasma membrane properties. In addition, we show that cancer cells present highly hydrophobic LD, compatible with a CE enrichment of these organelles. The distinct biophysical properties of LD contribute to the diversity of these organelles, suggesting that the specific alterations in their properties might be one of the mechanisms triggering LD pathophysiological actions and/or be related to the different mechanisms underlying LD metabolism.


Assuntos
Lauratos , Gotículas Lipídicas , Gotículas Lipídicas/metabolismo , Lauratos/análise , Lauratos/metabolismo , Metabolismo dos Lipídeos , 2-Naftilamina/análise , 2-Naftilamina/metabolismo
8.
FEBS J ; 290(13): 3383-3399, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-36808692

RESUMO

Acid-ß-glucosidase (GCase, EC3.2.1.45), the lysosomal enzyme which hydrolyzes the simple glycosphingolipid, glucosylceramide (GlcCer), is encoded by the GBA1 gene. Biallelic mutations in GBA1 cause the human inherited metabolic disorder, Gaucher disease (GD), in which GlcCer accumulates, while heterozygous GBA1 mutations are the highest genetic risk factor for Parkinson's disease (PD). Recombinant GCase (e.g., Cerezyme® ) is produced for use in enzyme replacement therapy for GD and is largely successful in relieving disease symptoms, except for the neurological symptoms observed in a subset of patients. As a first step toward developing an alternative to the recombinant human enzymes used to treat GD, we applied the PROSS stability-design algorithm to generate GCase variants with enhanced stability. One of the designs, containing 55 mutations compared to wild-type human GCase, exhibits improved secretion and thermal stability. Furthermore, the design has higher enzymatic activity than the clinically used human enzyme when incorporated into an AAV vector, resulting in a larger decrease in the accumulation of lipid substrates in cultured cells. Based on stability-design calculations, we also developed a machine learning-based approach to distinguish benign from deleterious (i.e., disease-causing) GBA1 mutations. This approach gave remarkably accurate predictions of the enzymatic activity of single-nucleotide polymorphisms in the GBA1 gene that are not currently associated with GD or PD. This latter approach could be applied to other diseases to determine risk factors in patients carrying rare mutations.


Assuntos
Celulases , Doença de Gaucher , Doença de Parkinson , Humanos , Doença de Gaucher/tratamento farmacológico , Doença de Gaucher/genética , Doença de Parkinson/genética , Heterozigoto , Mutação , Celulases/genética
9.
J Photochem Photobiol B ; 228: 112404, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-35196617

RESUMO

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.


Assuntos
Análise de Dados , Corantes Fluorescentes , 2-Naftilamina/análogos & derivados , 2-Naftilamina/química , Técnicas de Cultura de Células , Membrana Celular/metabolismo , Polarização de Fluorescência , Corantes Fluorescentes/química , Lauratos
10.
Artigo em Inglês | MEDLINE | ID: mdl-33892149

RESUMO

Niemann-Pick disease type C (NPC) is a complex and rare pathology, which is mainly associated to mutations in the NPC1 gene. This disease is phenotypically characterized by the abnormal accumulation of multiple lipid species in the acidic compartments of the cell. Due to the complexity of stored material, a clear molecular mechanism explaining NPC pathophysiology is still not established. Abnormal sphingosine accumulation was suggested as the primary factor involved in the development of NPC, followed by the accumulation of other lipid species. To provide additional mechanistic insight into the role of sphingosine in NPC development, fluorescence spectroscopy and microscopy were used to study the biophysical properties of biological membranes using different cellular models of NPC. Addition of sphingosine to healthy CHO-K1 cells, in conditions where other lipid species are not yet accumulated, caused a rapid decrease in plasma membrane and lysosome membrane fluidity, suggesting a direct effect of sphingosine rather than a downstream event. Changes in membrane fluidity caused by addition of sphingosine were partially sustained upon impaired trafficking and metabolization of cholesterol in these cells, and could recapitulate the decrease in membrane fluidity observed in NPC1 null Chinese Hamster Ovary (CHO) cells (CHO-M12) and in cells with pharmacologically induced NPC phenotype (treated with U18666A). In summary, these results show for the first time that the fluidity of the membranes is altered in models of NPC and that these changes are in part caused by sphingosine, supporting the role of this lipid in the pathophysiology of NPC.


Assuntos
Doença de Niemann-Pick Tipo C/patologia , Esfingolipídeos/metabolismo , Esfingosina/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Endossomos/metabolismo , Lisossomos/metabolismo , Doença de Niemann-Pick Tipo C/metabolismo , Fenótipo
11.
Elife ; 92020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32167466

RESUMO

Every cell is protected by a semipermeable membrane. Peptides with the right properties, for example Antimicrobial peptides (AMPs), can disrupt this protective barrier by formation of leaky pores. Unfortunately, matching peptide properties with their ability to selectively form pores in bacterial membranes remains elusive. In particular, the proline/glycine kink in helical peptides was reported to both increase and decrease antimicrobial activity. We used computer simulations and fluorescence experiments to show that a kink in helices affects the formation of membrane pores by stabilizing toroidal pores but disrupting barrel-stave pores. The position of the proline/glycine kink in the sequence further controls the specific structure of toroidal pore. Moreover, we demonstrate that two helical peptides can form a kink-like connection with similar behavior as one long helical peptide with a kink. The provided molecular-level insight can be utilized for design and modification of pore-forming antibacterial peptides or toxins.


Assuntos
Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Membrana Celular/metabolismo , Porinas/química , Porinas/metabolismo , Conformação Proteica , Membrana Celular/química , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Modelos Moleculares , Método de Monte Carlo , Relação Estrutura-Atividade
12.
Sci Rep ; 10(1): 3087, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-32080270

RESUMO

We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.


Assuntos
Fusão de Membrana , Microscopia de Fluorescência/métodos , Peptídeos/química , Lipossomas Unilamelares/química , Colesterol/química , Cor , Dimerização , Transferência Ressonante de Energia de Fluorescência , Lipídeos/química , Lipopeptídeos/química , Microscopia Confocal , Polissorbatos/química , Espectrometria de Fluorescência
13.
ACS Nano ; 12(1): 813-819, 2018 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-29244483

RESUMO

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.


Assuntos
Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Lipossomos/metabolismo , Permeabilidade da Membrana Celular , Microscopia de Fluorescência , Tamanho da Partícula , Permeabilidade , Espectrometria de Fluorescência
14.
Nanoscale ; 10(40): 19064-19073, 2018 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-30288507

RESUMO

A model system for membrane fusion, inspired by SNARE proteins and based on two complementary lipopeptides CPnE4 and CPnK4, has been recently developed. It consists of cholesterol (C), a poly(ethylene glycol) linker (Pn) and either a cationic peptide K4 (KIAALKE)4 or an anionic peptide E4 (EIAALEK)4. In this paper, fluorescence spectroscopy is used to decipher distinct but complementary roles of these lipopeptides during early stages of membrane fusion. Molecular evidence is provided that different distances of E4 in CPnE4 and K4 in CPnK4 from the bilayer represent an important mechanism, which enables fusion. Whereas E4 is exposed to the bulk and solely promotes membrane binding of CPnK4, K4 loops back to the lipid-water interface where it fulfills two distinct roles: it initiates bilayer contact by binding to CPnE4 containing bilayers; and it initiates fusion by modulating the bilayer properties. The interaction between CPnE4 and CPnK4 is severely down-regulated by binding of K4 to the bilayer and possible only if the lipopeptides approach each other as constituents of different bilayers. When the complementary lipopeptides are localized in the same bilayer, hetero-coiling is disabled. These data provide crucial insights as to how fusion is initiated and highlight the importance of both peptides in this process.


Assuntos
Materiais Biomiméticos/química , Bicamadas Lipídicas/química , Lipopeptídeos/química , Fusão de Membrana , Proteínas SNARE/química , Animais , Humanos
15.
Sci Rep ; 6: 38035, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905555

RESUMO

Understanding interactions of calcium with lipid membranes at the molecular level is of great importance in light of their involvement in calcium signaling, association of proteins with cellular membranes, and membrane fusion. We quantify these interactions in detail by employing a combination of spectroscopic methods with atomistic molecular dynamics simulations. Namely, time-resolved fluorescent spectroscopy of lipid vesicles and vibrational sum frequency spectroscopy of lipid monolayers are used to characterize local binding sites of calcium in zwitterionic and anionic model lipid assemblies, while dynamic light scattering and zeta potential measurements are employed for macroscopic characterization of lipid vesicles in calcium-containing environments. To gain additional atomic-level information, the experiments are complemented by molecular simulations that utilize an accurate force field for calcium ions with scaled charges effectively accounting for electronic polarization effects. We demonstrate that lipid membranes have substantial calcium-binding capacity, with several types of binding sites present. Significantly, the binding mode depends on calcium concentration with important implications for calcium buffering, synaptic plasticity, and protein-membrane association.


Assuntos
Cálcio/metabolismo , Bicamadas Lipídicas/química , Fosfolipídeos/química , Sítios de Ligação , Sinalização do Cálcio , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Lipossomos/química , Lipossomos/metabolismo , Modelos Moleculares , Simulação de Dinâmica Molecular , Fosfolipídeos/metabolismo
16.
Faraday Discuss ; 160: 341-58; discussion 389-403, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23795509

RESUMO

The influence of monovalent salts (NaF, NaCl, NaBr, NaClO4, KCl) on the properties of lipid bilayers composed of binary mixtures of zwitterionic DOPC (dioleoylphosphatidylcholine) and cationic DOTAP (dioleoyltrimethylammoniumpropane) is experimentally measured and numerically simulated. Both approaches report a specific adsorption of the studied anions at the cationic bilayer. The adsorption is enhanced for higher content of DOTAP in DOPC/DOTAP mixtures and for larger anions (Br and ClO4-). The nonmonotonic dependence of the lipid headgroup mobility, determined using time-dependent fluorescence shifts of Laurdan located at the bilayer carbonyl level, on the content of cationic lipid is preserved in all examined salt solutions. Its maximum, however, is shifted towards higher DOTAP concentrations in the row: NaF < NaCl < NaBr. The same ordering of salts is found for the simulated area per lipid and the measured rigidification of pure DOTAP bilayers. Simulations reveal that Br strongly binds to the cationic headgroups of DOTAP neutralising the bilayer, which induces lateral inhomogeneities in the form of hydrophilic and hydrophobic patches at the membrane-water interface for pure DOTAP. In the equimolar DOPC/DOTAP mixture the neutralising effect of Br results in bending of the PC headgroups to a bilayer-parallel orientation. F-, while attracted to the DOTAP bilayer, has an opposite effect to that of Br-, i.e. it increases local mobility at the lipid carbonyl level. We attribute this effect to the disruption of the hydrogen-bonded structure of the molecules of lipids and water caused by the presence of the adsorbed F-.


Assuntos
Cátions , Bicamadas Lipídicas , Sais/química , Simulação de Dinâmica Molecular , Espectrometria de Fluorescência
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA