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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 K D is ∼0.8 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. • Grb2 is predominantly found in its auto-inhibited form (dimer) in solution. • Grb2 Y160F exhibits a monomer-dimer equilibrium that favors the monomer in solution. • Grb2 Y160F presents a slingshot like mechanism that induces dimer dissociation. • SAXS and MD were combined to determine the Grb2 Y160F monomer ensemble in solution. • The ability of Grb2 Y160F to dimerize suggests distinct dimerization interfaces. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Conformational changes of the cyclic (Lo) peptide Labaditin (VWTVWGTIAG) and its linear analogue (L 1 ) 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 L 1 interacts with SDS micelles to a higher extent than does Lo. Further, the MD simulation demonstrated that both Lo and L 1 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, L 1 displays a random-coil structure in aqueous medium, pH 7.0, and it acquires a β -structure upon interaction with SDS and LPC, albeit with structural differences in each medium. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

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 (AlPcS 3) 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. • Light induced lipid peroxidation and membrane protection by D-PUFA and α-tocopherol were investigated. • Trisulfonated aluminum phthalocyanine damaged membranes by type I and type II photosensitized oxidation mechanisms. • The presence of D-PUFA strongly inhibits the photoinduced lipid oxidation. • α-tocopherol augment the protective effects of D-PUFAs. • In certain conditions, α-tocopherol increases membrane damage by the tocopherol-mediated peroxidation. [ABSTRACT FROM AUTHOR]

Abstract: We investigate the formation of ferrihydrite nanoparticles (NPs) by hydrolysis of the Fe(III) alkoxide Fe(OtBu)3. Controlled amounts of water, up to 3.0vol%, were added to the precursor solution yielding a series of hydrolyzed samples ranging from P0.0 (the unreacted precursor) to P3.0. X-ray diffraction (XRD) analysis evidenced the formation of high-crystalline ferrihydrite NP in sample P3.0, with grain size estimate of about 3.2nm. The transition from the molecular precursor to the formation of crystalline magnetic NPs was followed through magnetization measurements M(T) and M(H), as well as Mössbauer spectroscopy (MS). M(T) measurements indicate a paramagnetic (PM) behavior for sample P0.0, characteristic of binuclear Fe–O–Fe units, which evolves to a superparamagnetic (SPM) behavior, with an energy barrier for the blocking process estimated for sample P3.0 as E a=4.9×10−21 J (E a/k B=355K), resulting in a high effective anisotropy constant K eff=290kJ/m3. Magnetization loops at 5K progressively change from PM-like to ferromagnetic-like shape upon increasing the hydrolysis process, although hysteresis (H c≈500Oe) only is apparent for P2.0 and higher. MS spectra at room temperature are PM/SPM doublets for all samples, while the MS spectra at T=4.2K reveal increasingly well-defined magnetic ordering as hydrolysis of the precursor stepwise progresses until well-crystallized ferrihydrite particles are formed. [Copyright &y& Elsevier]

Abstract: Samples of and fine particles prepared by coprecipitation method have been studied by X-ray powder diffraction at room temperature. The oxygen position, the lattice parameter, the mean size of the nanoparticles and the cation distribution have been determined by means of Rietveld analysis, indicating the existence of mixed ferrites in both samples. These results are also evidenced through magnetic measurements in ferrofluid samples at room temperature. [Copyright &y& Elsevier]

Abstract: This work reports on the structural characterization of a copper ferrite nanoparticle sample, prepared by coprecipitation method, on both short (EXAFS) and long range (XRD) scales. The diffractograms obtained at room temperature were used for Rietveld refinement to determine the lattice parameters, the oxygen position, the mean size of the nanomaterial and the inversion degree, indicating the existence of a mixed ferrite. This cation distribution is also evidenced through both Cu and Fe EXAFS measurements at room temperature. [Copyright &y& Elsevier]