The thermal denaturation of azurin from Pseudomonas aeruginosa was investigated by means of differential scanning calorimetry (DSC), electron spin resonance (ESR), and optical density (OD) experiments, with the aim of determining its thermodynamic stability and the thermally induced conformational changes of its active site. DSC experiments have shown an irreversible and complex unfolding path. In order to characterize the kinetically controlled step, DSC measurements were carried out at different scan rates. An extrapolation of the experimental heat capacity data to infinite scan rate allowed all the kinetic and thermodynamic parameters related to the process to be obtained. All these parameters extracted from the calorimetric data were verified by means of a curve-fitting program using an equation containing all information necessary to fully describe the unfolding process in details. Thermal denaturation, followed up to 82 °C by ESR and OD measurements, allowed us to study the structural variations of the copper environment at different temperatures. The A/fu thermodynamic, together with the value of ACp calculated according to an approach taking into account the common features of protein unfolding and dissolution of hydrophobic compounds, was used to evaluate the thermodynamic stability (AG) for the reversible process over the entire temperature range of denaturation. The high value of the maximum stability thus calculated was explained by the stabilizing effect of copper.
The disulfide bond connecting Cys-3 and Cys-26 in wild type azurin has been removed to study the contribution of the -SS- bond to the high thermal resistance previously registered for this protein (. J. Phys. Chem. 99:14864-14870). Site-directed mutagenesis was used to replace both cysteines for alanines. The characterization of the Cys-3Ala/Cys-26Ala azurin mutant has been carried out by means of electron paramagnetic resonance spectroscopy at 77 K, UV-VIS optical absorption, fluorescence emission and circular dichroism at room temperature. The results show that the spectral features of the Cys-3Ala/Cys-26Ala azurin resemble those of the wild type azurin, indicating that the double mutation does not affect either the formation of the protein's overall structure or the assembly of the metal-binding site. The thermal unfolding of the Cys-3Ala/Cys-26Ala azurin has been followed by differential scanning calorimetry, optical absorption variation at lambda(max) = 625 nm, and fluorescence emission using 295 nm as excitation wavelength. The analysis of the data shows that the thermal transition from the native to the denaturated state of the modified azurin follows the same multistep unfolding pathway as observed in wild type azurin. However, the removal of the disulfide bridge results in a dramatic reduction of the thermodynamic stability of the protein. In fact, the transition temperatures registered by the different techniques are down-shifted by about 20 degrees C with respect to wild type azurin. Moreover, the Gibbs free energy value is about half of that found for the native azurin. These results suggest that the disulfide bridge is a structural element that significantly contributes to the high stability of wild type azurin.
Carnosine is an endogenously synthesized dipeptide composed of -alanine and L-histidine. It acts as a free radical scavenger and possesses antioxidant properties. Carnosine reduces proinflammatory and profibrotic cytokines such as transforming growth factor- (TGF-), IL-1, and TNF-␣ in different experimental settings. In the present study, we investigated the efficacy of carnosine on the animal model of bleomycin-induced lung injury. Mice were subjected to intratracheal administration of bleomycin and were assigned to receive carnosine daily by an oral bolus of 150 mg/kg. One week after fibrosis induction, bronchoalveolar lavage (BAL) cell counts and TGF- levels, lung histology, and immunohistochemical analyses for myeloperoxidase, TGF-, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase were performed. Finally, apoptosis was quantified by terminal deoxynucleotidyltransferase-mediated UTP end-labeling assay. After bleomycin administration, carnosine-treated mice exhibited a reduced degree of lung damage and inflammation compared with wild-type mice, as shown by the reduction of 1) body weight, 2) mortality rate, 3) lung infiltration by neutrophils (myeloperoxidase activity and BAL total and differential cell counts), 4) lung edema, 5) histological evidence of lung injury and collagen deposition, 6) lung myeloperoxidase, TGF-, inducible nitric oxide synthase, nitrotyrosine, and poly(ADP-ribose) polymerase immunostaining, 7) BAL TGF- levels, and 8) apoptosis. Our results indicate that orally administered carnosine is able to prevent bleomycin-induced lung injury likely through its direct antioxidant properties.
The mechanisms underlying the formation of extracellular amyloid plaques on neuronal membranes, a major hallmark of Alzheimer's disease, are the subject of intense debate. Here we use multiscale simulations and analytical theory to unveil the early steps of the spontaneous self-assembly of membrane-embedded α-helical Aβ (1-40) peptides. Based on a simple analytical model describing the electrostatic repulsions among water-exposed charged residues, the presence of distorted structures called "frustrated helices" is predicted. Large scale (20 μs) Coarse Grained simulations of 36 replicas of Aβ (1-40) performed within a POPC lipid matrix confirmed the formation of supramolecular assemblies which resemble a twisted ribbon. Fully atomistic simulations have demonstrated the stability of these helical structures. Concomitant to the formation of these large assemblies, CG simulations evidenced membrane curvature and substantiate the view that these assemblies may entail mechanical stress on membrane structure. We think that these findings provide an alternative view to the traditional models that consider a conformational transition towards β-sheet rich structures as a prerequisite for triggering membrane damage and, eventually, neurotoxicity.
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