RESUMO
The Growth factor receptor-bound protein 2 (Grb2) participates in early signaling complexes and regulates tyrosine kinase-mediated signal transduction through a monomer-dimer equilibrium. Grb2 dimeric state inhibits signal transduction whereas the monomer promotes signaling downstream. Since Grb2 dimer KD is â¼0.8 µM, studies focused on the monomer are still challenging and require mutations or interaction with phosphotyrosine peptides. However, these mutants were never characterized considering their effects on protein structure and dynamics in solution. Here, we present the biophysical characterization of Grb2Y160F, the first Grb2 mutant to induce protein monomerization without disrupting its native behavior in solution due to net charge modifications or interaction with peptides. We also identified that Grb2Y160F exists in a monomer-dimer equilibrium. Grb2Y160F ability to dimerize implies that different dimerization interfaces might regulate signaling pathways in distinct ways and raises an important question about the role of the Y160 residue in other dimerization interfaces.
RESUMO
Lipid peroxidation (LPO) plays a key role in many age-related neurodegenerative conditions and other disorders. Light irradiation can initiate LPO through various mechanisms and is of importance in retinal and dermatological pathologies. The introduction of deuterated polyunsaturated fatty acids (D-PUFA) into membrane lipids is a promising approach for protection against LPO. Here, we report the protective effects of D-PUFA against the photodynamically induced LPO, using illumination in the presence of the photosensitizer trisulfonated aluminum phthalocyanine (AlPcS3) in liposomes and giant unilamellar vesicles (GUV), as assessed in four experimental models: 1) sulforhodamine B leakage from liposomes, detected with fluorescence correlation spectroscopy (FCS); 2) formation of diene conjugates in liposomal membranes, measured by absorbance at 234 nm; 3) membrane leakage in GUV assessed by optical phase-contrast intensity observations; 4) UPLC-MS/MS method to detect oxidized linoleic acid (Lin)-derived metabolites. Specifically, in liposomes or GUV containing H-PUFA (dilinoleyl-sn-glycero-3-phosphatidylcholine), light irradiation led to an extensive oxidative damage to bilayers. By contrast, no damage was observed in lipid bilayers containing 20% or more D-PUFA (D2-Lin or D10-docosahexanenoic acid). Remarkably, addition of tocopherol increased the dye leakage from liposomes in H-PUFA bilayers compared to photoirradiation alone, signifying tocopherol's pro-oxidant properties. However, in the presence of D-PUFA the opposite effect was observed, whereby adding tocopherol increased the resistance to LPO. These findings suggest a method to augment the protective effects of D-PUFA, which are currently undergoing clinical trials in several neurological and retinal diseases that involve LPO.
Assuntos
Bicamadas Lipídicas , Espectrometria de Massas em Tandem , Cromatografia Líquida , Ácidos Graxos , Ácidos Graxos Insaturados/farmacologia , Peroxidação de Lipídeos , LipossomosRESUMO
Protein aggregation plays important roles in life science as, for instance, those associated to neurodegenerative diseases. Although extensive efforts have been done to elucidate all the possible variables related to the aggregation process, much has yet to be done to unveil the main pathways governing protein assembling. In the current work, we induce bovine serum albumin (BSA) association, at pH 3.7, by adding sodium dodecyl sulfate (SDS) and sodium perfluorooctanoate (SPFO) surfactants to BSA solution as promoters of protein aggregation. Firstly, we combine molecular dynamic simulations (MD) to obtain a partially unfolded state of BSA's monomer at the acid pH and small angle X-ray scattering (SAXS) to validate the model. Interestingly, we found by SAXS that at pH 3.7 BSA monomers coexist with dimers in surfactant-free solution. Upon SDS and SPFO addition, the partial unfolded BSA may evolve to large aggregates depending on surfactant concentration. The threshold occurs at 30:1 and 45:1 SDS:BSA and SPFO:BSA molar ratio, respectively, according to turbidity, Thioflavin (ThT) fluorescence, synchrotron radiation circular dichroism (SRCD), SAXS and scanning electron microscopy (SEM) experiments. BSA aggregates are larger in the presence of SDS and structurally more defined upon SPFO binding. Isothermal titration calorimetry (ITC) results give support to infer that both surfactants initially bind to the BSA macromolecule forming a complex. Then, these complexes self-associate towards supramolecular aggregates. Taking into account the physicochemical characteristics of both surfactants and also MD simulations we may suggest that the higher rigidity of the fluorinated chains in respect to hydrogenated ones is crucial to induce more ordered and smaller BSA's aggregates. Our results thus evidence that the ligand structural flexibility might be of a key importance in the pathway of protein aggregation and may pave the way to better understand the early steps of neurodegenerative disorders.
Assuntos
Simulação de Dinâmica Molecular , Soroalbumina Bovina/química , Tensoativos/química , Animais , Caprilatos/química , Bovinos , Fluorocarbonos/química , Halogenação , Hidrogenação , Tamanho da Partícula , Agregados Proteicos , Desdobramento de Proteína , Espalhamento a Baixo Ângulo , Dodecilsulfato de Sódio/química , Propriedades de Superfície , Difração de Raios XRESUMO
In the present work, the production of bioemulsifier (BE) by a lactic acid bacterium (LAB) grown at 25⯰C in lactic whey-based media for 24â¯h was evaluated. Maximum production was detected in a medium containing yeast extract, peptone and lactic whey (LAPLW medium), with a yield of 270â¯mgâ¯L-1. The BE proved to be more innocuous for Caco-2â¯cells, used as a toxicological indicator, than the non-ionic surfactant Triton X-100. In addition, the microbial product presented higher stability to changes in temperature (37⯰C to 100⯰C), pH (2-10), and salt concentration (5% and 20%, w/v) than the synthetic surfactant. Regarding emulsifying capacity tested against different hydrophobic substrates (kerosene, motor oil, diesel, sunflower oil, and grape oil), the BE displayed E24 values similar to or even better than those of Triton X-100. Finally, Triton X-100 caused irreversible modifications on the giant unilamellar vesicles (used as model membrane system), promoting the solubilization of the lipid bilayers. Nevertheless, BE induced temporary modifications of the membrane, which is associated with incorporation of the bioproduct in the outer layer. These results demonstrate the role of BE in biological processes, including reversible changes in microbial membranes to enhance the access to hydrophobic substrates.
Assuntos
Biotecnologia/métodos , Emulsificantes/isolamento & purificação , Enterococcus/metabolismo , Ácido Láctico/metabolismo , Soro do Leite/metabolismo , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Emulsificantes/metabolismo , Emulsificantes/toxicidade , Emulsões , Humanos , Interações Hidrofóbicas e Hidrofílicas , Octoxinol/química , Petróleo/metabolismo , Óleos de Plantas/metabolismo , TemperaturaRESUMO
We present molecular dynamics simulations of cardiolipin (CL) and CL monohydroperoxized derivative (CLOOH) monolayers to investigate the initial steps of phospholipid oxidation induced by methylene blue (MB) photoexcitation under continuous illumination. We considered different MB atomic charge distributions to simulate the MB electronic distribution in the singlet ground and triplet excited states. Simulation results allied to experimental data revealed that initial CL photooxidation probably occurs via a type II mechanism, to produce lipid hydroperoxide by singlet oxygen attack to the alkyl chain unsaturations. The resulting hydroperoxide group prefers to reside near the aqueous interface, to increase the membrane surface area and to decrease lipid packing. Interestingly, MB orientation changes from nearly parallel to the water-monolayer interface in the ground state to normal to the interface in its triplet excited state. The latter orientation favors oxidative chain reaction continuity via a type I mechanism, during which the hydrogen atom must be transferred from the hydroperoxide group to triplet MB. Taken together, the present results can be extrapolated to improve our understanding of how oxidation progresses in lipidic biomembrane, which will lead to the formation of oxidized species with shortened chains and will cause severe photodamage to self-organized systems.
Assuntos
Cardiolipinas/química , Peróxidos Lipídicos/química , Membranas Artificiais , Azul de Metileno/química , Azul de Metileno/efeitos da radiação , Luz , Peroxidação de Lipídeos , Simulação de Dinâmica Molecular , Oxigênio Singlete/químicaRESUMO
Antimicrobial peptides (AMPs) are cationic peptides that kill bacteria with a broad spectrum of action, low toxicity to mammalian cells and exceptionally low rates of bacterial resistance. These features have led to considerable efforts in developing AMPs as an alternative antibacterial therapy. In vitro studies have shown that AMPs interfere with membrane bilayer integrity via several possible mechanisms, which are not entirely understood. We have performed the synthesis, membrane lysis measurements, and biophysical characterization of a novel hybrid peptide. These measurements show that PA-Pln149 does not form nanopores, but instead promotes membrane rupture. It causes fast rupture of the bacterial model membrane (POPG-rich) at concentrations 100-fold lower than that required for the disruption of mammalian model membranes (POPC-rich). Atomistic molecular dynamics (MD) simulations were performed for single and multiple copies of PA-Pln149 in the presence of mixed and pure POPC/POPG bilayers to investigate the concentration-dependent membrane disruption by the hybrid peptide. These simulations reproduced the experimental trend and provided a potential mechanism of action for PA-Pln149. It shows that the PA-Pln149 does not form nanopores, but instead promotes membrane destabilization through peptide aggregation and induction of membrane negative curvature with the collapse of the lamellar arrangement. The sequence of events depicted for PA-Pln149 may offer insights into the mechanism of action of AMPs previously shown to induce negative deformation of membrane curvature and often associated with peptide translocation via non-bilayer intermediate structures.
RESUMO
Biological membranes and model lipid systems containing high amounts of unsaturated lipids and sterols are subject to chemical and/or photo-induced lipid oxidation, which leads to the creation of exotic oxidized lipid products (OxPLs). OxPLs are known to have significant physiological impact in cellular systems and also affect physical properties of both biological and model lipid bilayers. In this paper we (i) provide a perspective on the existing literature on simulations of lipid bilayer systems containing oxidized lipid species as well as the main related experimental results, (ii) describe our new data of all-atom and coarse-grained simulations of hydroperoxidized lipid monolayer and bilayer systems and (iii) provide a comparison of the MARTINI and ELBA coarse grained force fields for lipid bilayer systems. We show that the better electrostatic treatment of interactions in ELBA is able to resolve previous conflicts between experiments and simulations. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.
Assuntos
Bicamadas Lipídicas/química , Lipídeos/química , Simulação de Dinâmica Molecular , Oxirredução , Eletricidade EstáticaRESUMO
Conformational changes of the cyclic (Lo) peptide Labaditin (VWTVWGTIAG) and its linear analogue (L1) promoted by presence of anionic sodium dodecyl sulfate (SDS) and zwitterionic L-α-Lysophosphatidylcholine (LPC) micelles were investigated. Results from λ(max) blue-shift of tryptophan fluorescence emission combined with Stern-Volmer constants values and molecular dynamics (MD) simulations indicated that L1 interacts with SDS micelles to a higher extent than does Lo. Further, the MD simulation demonstrated that both Lo and L1 interact similarly with LPC micelles, being preferentially located at the micelle/water interface. The peptide-micelle interaction elicits conformational changes in the peptides. Lo undergoes limited modifications and presents unordered structure in both LPC and SDS micelles. On the other hand, L1 displays a random-coil structure in aqueous medium, pH 7.0, and it acquires a ß-structure upon interaction with SDS and LPC, albeit with structural differences in each medium.
Assuntos
Micelas , Peptídeos Cíclicos/química , Peptídeos/química , Ânions/química , Dicroísmo Circular , Simulação de Dinâmica MolecularAssuntos
Toxicologia , Toxicologia , Venenos , Intoxicação , Toxinas Biológicas , Toxicologia , Bioquímica , FarmacologiaRESUMO
Using phase contrast and fluorescence microscopy we study the influence of the alkylphospholipid, ALP, 10-(octyloxy) decyl-2-(trimethylammonium) ethyl phosphate, ODPC, in giant unilamellar vesicles, GUVs, composed of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), brain sphingomyelin (SM) and cholesterol (Chol). The results show that adding 100µM ODPC (below CMC) to the outer solution of GUVs promotes DOPC membrane disruption over a period of 1h of continuous observation. On the other hand, the presence of SM and Chol in homogeneous fluid lipid bilayers protects the membrane from disruption. Interestingly, by adding 100µM ODPC to GUVs containing DOPC:SM:Chol (1:1:1), which display liquid ordered (Lo)-liquid disordered (Ld) phase coexistence, the domains rapidly disappear in less than 1min of ODPC contact with the membrane. The lipids are subsequently redistributed to liquid domains within a time course of 14-18min, reflecting that the homogenous phase was not thermodynamically stable, followed by rupture of the GUVs. A similar mechanism of action is also observed for perifosine, although to a larger extent. Therefore, the initial stage of lipid raft disruption by both ODPC and perifosine, and maybe other ALPS, by promoting lipid mixing, may be correlated with their toxicity upon neoplastic cells, since selective (dis)association of essential proteins within lipid raft microdomains must take place in the plasma membrane.
Assuntos
Glicerofosfolipídeos/química , Bicamadas Lipídicas/química , Lipídeos de Membrana/química , Microdomínios da Membrana/química , Lipossomas Unilamelares/química , Colesterol/química , Fluidez de Membrana , Microscopia de Fluorescência , Microscopia de Contraste de Fase , Modelos Químicos , Modelos Moleculares , Fosfatidilcolinas/química , Fosforilcolina/análogos & derivados , Fosforilcolina/química , Esfingomielinas/química , TermodinâmicaRESUMO
The aggregate morphology of meso-tetrakis(4-sulfonatophenyl) porphyrin (TPPS(4)) in aqueous solution is investigated by using small angle x-ray scattering (SAXS) technique. Measurements were performed at pH 4.0 and 9.0 to monitor the pH influence on the structural parameters of the aggregates. Radii of gyration were obtained from distance distribution functions p(r) analysis. The experimental data of TPPS(4) at pH 4.0 showed well-defined oscillations characteristic of large aggregates in contrast to the SAXS curve of 5 mM TPPS(4) at pH 9.0, where both a significant decrease in the intensity and the disappearance of the oscillation peaks suggest the dissociation of the aggregate. A 340-A long "hollow" cylinder with shell thickness of 20 A, compatible to the porphyrin molecule dimension, represents well the scattering curve of the aggregates at pH 4.0. According to the fitting parameters, 26 porphyrin molecules self-associate into a ringlike configuration in the plane of the cylinder cross-section. The total number of porphyrin molecules in the whole aggregate was also estimated as approximately 3000. The model compatible to SAXS data of a hollow cylinder with J-aggregation in the cross-section and H-aggregation (columnar stacking) between the cylinder layers is consistent with optical absorption spectroscopic data both in the literature and obtained in this work.
Assuntos
Cristalização/métodos , Cristalografia por Raios X/métodos , Modelos Moleculares , Porfirinas/química , Água/química , Simulação por Computador , Concentração de Íons de Hidrogênio , Substâncias Macromoleculares , Conformação Molecular , Polímeros/síntese química , Porfirinas/síntese química , SoluçõesRESUMO
The ferricytochrome-c (cyt-c) shows a complex unfolding pathway characterized by a series of stable partially folded states. When titrated with HCl at low ionic strength, two transitions are detected. At pH 2, cyt-c assumes the U1 unfolded state, whereas the successive addition of Cl(-) ion from either HCl or NaCl induces the recompaction to a molten globule conformation (A1 and A2 states, respectively). A second unfolded state (U2) is also observed at pH 12. Recent data evidence different features for the local structure of the heme in the different states. To derive relationships between local and overall conformations, we analyzed the structural characteristics of the different states by synchrotron small angle X-ray scattering. The results show that in the acidic-unfolded U1 form the protein assumes a worm-like conformation, whereas in the alkaline-unfolded U2 state, the cyt-c is globular. Moreover, the molten globule states induced by adding HCl or NaCl to U1 appear structurally different: in the A1 state cyt-c is dimeric and less compact, whereas in the A2 form the protein reverts to a globular-like conformation. According to the local heme structure, a molecular model for the different forms is derived.
Assuntos
Grupo dos Citocromos c/química , Espalhamento de Radiação , Animais , Dicroísmo Circular , Dimerização , Cavalos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Método de Monte Carlo , Miocárdio/metabolismo , Conformação Proteica , Desnaturação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Espectrofotometria , Raios XRESUMO
The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.
Assuntos
Anestésicos Locais/química , Micelas , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Espalhamento de Radiação , Espectrometria de Fluorescência , Marcadores de Spin , Tetracaína/químicaRESUMO
Lipophilic guanosine derivatives act as self-assembled ionophores. In the presence of alkali metal ions in organic solvents, these G derivatives can form tubular polymeric structures. The molecular aggregates formed by 3',5'-didecanoyl-2'-deoxyguanosine (1) have been characterised by SANS and NMR spectroscopy. The polymer is structured as a pile of stacked G quartets held together by the alkali metal ions that occupy the column's central channel. The deoxyribose moieties, with their alkyl substituents, surround the stacked G quartets, and the nucleoside's long-chain alkyl tails are in intimate contact with the organic solvent. In this polymeric structure, there is an amazing regularity in the rotamers around the glycosidic bond within each G quartet and in the repeat sequence of the G quartets along the columns. In hydrocarbon solvents, these columnar aggregates form lyomesophases of the cholesteric and hexagonal types.
Assuntos
Guanosina/química , Conformação de Ácido Nucleico , Biopolímeros/química , Espectroscopia de Ressonância MagnéticaRESUMO
The variable-period x-ray standing wave (XSW) technique is emerging as a powerful tool for studying membrane structure. However, two significant problems arise when the method is used to characterize membranes of thickness dL < 100 A. First, the surface roughness, sigma(r), of the supporting reflecting mirror convolutes with the intrinsic half-width of the marker atom distribution in the membrane, sigma(in), and contributes to an apparent half-width, sigma, which is measured in the XSW experiment. Here we show how the latter terms are related quantitatively [sigma(in) = (sigma2 - sigma(r)2)(1/2)], such that rough mirrors give rise to larger marker atom distribution widths, sigma, and how the required quantity sigma(in) can be determined in the XSW measurement. Second, when the mean position of the marker atom layer, (z), is close to one or both boundaries of the membrane, its distribution function is truncated at the boundary. In such cases, we show why marker atom distribution should be expressed in terms of its first and second moments. We also demonstrate by numerical simulations of realistic samples how the physical parameters, sigma(r), sigma, (z), and dL, affect x-ray reflectivity and fluorescence yield profiles as an aid in their interpretation.
Assuntos
Membrana Celular/química , Fenômenos Biofísicos , Biofísica , Fenômenos Eletromagnéticos , Fluorescência , Lipídeos de Membrana/química , Proteínas de Membrana/química , Modelos Químicos , Espalhamento de Radiação , Propriedades de Superfície , Raios XRESUMO
A series of model membranes as Langmuir-Blodgett (LB) films composed of long-chain zinc alkanoates (saturated fatty acid salts) was used to evaluate the spatial resolution of the variable-period x-ray standing-wave (XSW) technique. The chain length dependence of the zinc mean position (z) above the supporting substrate demonstrates that it is possible to detect differences in (z) of 1-2 A. Thus 1-2 A is the spatial resolution of the method in the current application. The data show that the chain tilt angle is chain length dependent, varying from 40 degrees to 0 degrees for alkanoates 18 and 24 carbon atoms long, respectively. The spread about the mean position of the zinc in the film, sigma(in), was found to be independent of chain length at 10.0 A for all members of the series. Sigma(in) was shown to be insensitive to the presence of a "spacer" omega-tricosenoic acid (omegaTA) bilayer placed between the zinc alkanoate LB film and the coated gold mirror. However, an overlayer of omegaTA sharpened the zinc ion distribution and lowered the chain tilt angle. This study provides important information regarding sample composition and constitution that facilitates membrane structure determination by XSWs.