RESUMEN
The need to combat antimicrobial resistance is becoming more and more pressing. Here we investigate the working mechanism of a small cationic agent, N-alkylamide 3d, by conventional and high-speed atomic force microscopy. We show that N-alkylamide 3d interacts with the membrane of Staphylococcus aureus, where it changes the organization and dynamics of lipid domains. After this initial step, supramolecular structures of the antimicrobial agent attach on top of the affected membrane gradually, covering it entirely. These results demonstrate that lateral domains in the bacterial membranes might be affected by small antimicrobial agents more often than anticipated. At the same time, we show a new dual-step activity of N-alkylamide 3d that not only destroys the lateral membrane organization but also effectively covers the whole membrane with aggregates. This final step could render the membrane inaccessible from the outside and possibly prevent signaling and waste disposal of living bacteria.
RESUMEN
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
We employed all-atom MD simulations to investigate the impact of palmitoylation on the PAG transmembrane peptide within various lipid environments, including the less explored boundary region separating lipid-ordered (Lo) and lipid-disordered (Ld) membrane phases. We found that palmitoylation of the peptide reduces its impact on membrane thickness, particularly within the Lo and boundary environments. Despite their hydrophobic nature, the palmitoyl chains on the peptide did not significantly affect the hydration of the surrounding membrane. Interestingly, the boundary membrane environment was found to be especially compatible with the palmitoylated peptide, suggesting its potential for accumulation in phase boundaries. Our findings highlight the importance of understanding how palmitoylation-modified peptides behave within membranes, with crucial implications for cell signaling and membrane organization. This knowledge may also inform the optimization of lipid membrane-based drug delivery systems, by improving our understanding of how drugs and excipients can be most effectively arranged within these carriers.
Asunto(s)
Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Membrana Dobles de Lípidos/química , Lipoilación , Péptidos/metabolismoRESUMEN
Antimicrobial resistance is one of the leading concerns in medical care. Here we study the mechanism of action of an antimicrobial cationic tripeptide, AMC-109, by combining high speed-atomic force microscopy, molecular dynamics, fluorescence assays, and lipidomic analysis. We show that AMC-109 activity on negatively charged membranes derived from Staphylococcus aureus consists of two crucial steps. First, AMC-109 self-assembles into stable aggregates consisting of a hydrophobic core and a cationic surface, with specificity for negatively charged membranes. Second, upon incorporation into the membrane, individual peptides insert into the outer monolayer, affecting lateral membrane organization and dissolving membrane nanodomains, without forming pores. We propose that membrane domain dissolution triggered by AMC-109 may affect crucial functions such as protein sorting and cell wall synthesis. Our results indicate that the AMC-109 mode of action resembles that of the disinfectant benzalkonium chloride (BAK), but with enhanced selectivity for bacterial membranes.
Asunto(s)
Antiinfecciosos , Peptidomiméticos , Peptidomiméticos/farmacología , Peptidomiméticos/metabolismo , Péptidos Catiónicos Antimicrobianos/química , Antiinfecciosos/química , Staphylococcus aureus , Simulación de Dinámica Molecular , Membrana Celular/metabolismo , Antibacterianos/química , Pruebas de Sensibilidad MicrobianaRESUMEN
Antibiotics that use novel mechanisms are needed to combat antimicrobial resistance1-3. Teixobactin4 represents a new class of antibiotics with a unique chemical scaffold and lack of detectable resistance. Teixobactin targets lipid II, a precursor of peptidoglycan5. Here we unravel the mechanism of teixobactin at the atomic level using a combination of solid-state NMR, microscopy, in vivo assays and molecular dynamics simulations. The unique enduracididine C-terminal headgroup of teixobactin specifically binds to the pyrophosphate-sugar moiety of lipid II, whereas the N terminus coordinates the pyrophosphate of another lipid II molecule. This configuration favours the formation of a ß-sheet of teixobactins bound to the target, creating a supramolecular fibrillar structure. Specific binding to the conserved pyrophosphate-sugar moiety accounts for the lack of resistance to teixobactin4. The supramolecular structure compromises membrane integrity. Atomic force microscopy and molecular dynamics simulations show that the supramolecular structure displaces phospholipids, thinning the membrane. The long hydrophobic tails of lipid II concentrated within the supramolecular structure apparently contribute to membrane disruption. Teixobactin hijacks lipid II to help destroy the membrane. Known membrane-acting antibiotics also damage human cells, producing undesirable side effects. Teixobactin damages only membranes that contain lipid II, which is absent in eukaryotes, elegantly resolving the toxicity problem. The two-pronged action against cell wall synthesis and cytoplasmic membrane produces a highly effective compound targeting the bacterial cell envelope. Structural knowledge of the mechanism of teixobactin will enable the rational design of improved drug candidates.
Asunto(s)
Antibacterianos , Bacterias , Membrana Celular , Depsipéptidos , Viabilidad Microbiana , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/citología , Bacterias/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Pared Celular/metabolismo , Depsipéptidos/química , Depsipéptidos/farmacología , Difosfatos/química , Farmacorresistencia Bacteriana/efectos de los fármacos , Humanos , Lípidos/química , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Pirrolidinas/química , Azúcares/químicaRESUMEN
Molecularly thin, nanoporous thin films are of paramount importance in material sciences. Their use in a wide range of applications requires control over their chemical functionalities, which is difficult to achieve using current production methods. Here, the small polycyclic aromatic hydrocarbon decacyclene is used to form molecular thin films, without requiring covalent crosslinking of any kind. The 2.5 nm thin films are mechanically stable, able to be free-standing over micrometer distances, held together solely by supramolecular interactions. Using a combination of computational chemistry and microscopic imaging techniques, thin films are studied on both a molecular and microscopic scale. Their mechanical strength is quantified using AFM nanoindentation, showing their capability of withstanding a point load of 26 ± 9 nN, when freely spanning over a 1 µm aperture, with a corresponding Young's modulus of 6 ± 4 GPa. Our thin films constitute free-standing, non-covalent thin films based on a small PAH.
RESUMEN
The tear film at the ocular surface is covered by a thin layer of lipids. This oily phase stabilizes the film by decreasing its surface tension and improving its viscoelastic properties. Clinically, destabilization and rupture of the tear film are related to dry eye disease and are accompanied by changes in the quality and quantity of tear film lipids. In dry eye, eye drops containing oil-in-water emulsions are used for the supplementation of lipids and surface-active components to the tear film. We explore in detail the biophysical aspects of interactions of specific surface-active compounds, cetalkonium chloride and poloxamer 188, which are present in oil-in-water emulsions, with tear lipids. The aim is to better understand the macroscopically observed eye drops-tear film interactions by rationalizing them at the molecular level. To this end, we employ a multi-scale approach combining experiments on human meibomian lipid extracts, measurements using synthetic lipid films, and in silico molecular dynamics simulations. By combining these methods, we demonstrate that the studied compounds specifically interact with the tear lipid film enhancing its structure, surfactant properties, and elasticity. The observed effects are cooperative and can be further modulated by material packing at the tear-air interface.
Asunto(s)
Lípidos/química , Microscopía Fluorescente/métodos , Simulación de Dinámica Molecular , Películas Cinematográficas , Alcoholes Grasos/química , Humanos , Glándulas Tarsales/metabolismo , Modelos Teóricos , Poloxámero/química , Compuestos de Amonio Cuaternario/químicaRESUMEN
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.
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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
Adsorption of metal cations onto a cellular membrane changes its properties, such as interactions with charged moieties or the propensity for membrane fusion. It is, however, unclear whether cells can regulate ion adsorption and the related functions via locally adjusting their membrane composition. We employed fluorescence techniques and computer simulations to determine how the presence of cholesterol-a key molecule inducing membrane heterogeneity-affects the adsorption of sodium and calcium onto zwitterionic phosphatidylcholine bilayers. We found that the transient adsorption of sodium is dependent on the number of phosphatidylcholine head groups, while the strong surface binding of calcium is determined by the available surface area of the membrane. Cholesterol thus does not affect sodium adsorption and only plays an indirect role in modulating the adsorption of calcium by increasing the total surface area of the membrane. These observations also indicate how lateral lipid heterogeneity can regulate various ion-induced processes including adsorption of peripheral proteins, nanoparticles, and other molecules onto membranes.
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Calcio/química , Colesterol/química , Fosfatidilcolinas/química , Sodio/química , Simulación por Computador , Membranas Artificiales , Modelos Químicos , Imagen ÓpticaRESUMEN
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.
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Calcio/metabolismo , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Sitios de Unión , Señalización del Calcio , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Liposomas/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Fosfolípidos/metabolismoRESUMEN
Determination of the saccharide structure in solution is a laborious process that can be significantly enhanced by optical spectroscopies. Raman optical activity (ROA) spectra are particularly sensitive to the chirality and conformation. However, the interpretation of them is largely dependent on computational tools providing a limited precision only. To understand the limitations and the link between spectral shapes and the structure, in the present study we measured and interpreted using a combination of molecular dynamics (MD) and density functional theory (DFT) Raman and ROA spectra of glucose and mannose solutions. Factors important for analyses of mixtures of conformers, anomers, and different monosaccharides are discussed as well. The accuracy of the simulations was found to be strongly dependent on the quality of the hydration model; the dielectric continuum solvent model provided lower accuracy than averaging of many solvent-solute clusters. This was due to different conformer weighting rather than direct involvement of water molecules in scattering recorded as ROA. However, the cluster-based simulations also failed to correctly reproduce the ratios of principal monosaccharide forms. The best results were obtained by a combined MD/DFT simulation, with the ratio of α- and ß-anomers and the -CH2OH group rotamers determined experimentally by NMR. Then a decomposition of experimental spectra into calculated subspectra provided realistic results even for the glucose and mannose mixtures. Raman spectra decomposition provided a better overall accuracy (â¼5%) than ROA (â¼10%). The combination of vibrational spectroscopy with theoretical simulations represents a powerful tool for analysing the saccharide structure. Conversely, the ROA and Raman data can be used to verify the quality of MD force fields and other parameters of computational modeling.
RESUMEN
Since its introduction in 1998, genetically encoded pH-sensitive sensor ratiometric pHluorin proved to be a valuable tool for cell physiology studies. Here, we show how the sensitivity of pHluorin-based monitoring of intracellular pH changes performed with cell suspensions can be enhanced by using synchronously scanned fluorescence spectroscopy. In the suspensions of S. cerevisiae cells subjected to varying extracellular pH values, we have been able to measure statistically significant changes in intracellular pH of less than 0.1 unit, which were not detectable using a standard ratiometric approach.