Ligand-Induced structural changes in TEM‑1 probed by molecular dynamics and relative binding free energy calculations

The TEM family of enzymes has had a crucial impact on the pharmaceutical industry due to their important role in antibiotic resistance. Even with the latest technologies in structural biology and genomics, no 3D structure of a TEM- 1/antibiotic complex is known previous to acylation. Therefore, the comprehension of their capability in acylate antibiotics is based on the protein macromolecular structure uncomplexed. In this work, molecular docking, molecular dynamic simulations, and relative free energy calculations were applied in order to get a comprehensive and thorough analysis of TEM-1/ampicillin and TEM-1/amoxicillin complexes. We described the complexes and analyzed the effect of ligand binding on the overall structure. We clearly demonstrate that the key residues involved in the stability of the ligand (hot-spots) vary with the nature of the ligand. Structural effects such as (i) the distances between interfacial residues (Ser70−Oγ and Lys73−Nζ, Lys73−Nζ and Ser130−Oγ, and Ser70−Oγ−Ser130−Oγ), (ii) side chain rotamer variation (Tyr105 and Glu240), and (iii) the presence of conserved waters can be also influenced by ligand binding. This study supports the hypothesis that TEM-1 suffers structural modifications upon ligand binding.

Biochemical and structural characterization of β-lactamases tem-180 and tem-201

With the constant development of new antibiotics, selective pressure is a force to reckon when investigating antibiotic resistance. Although advantageous for medical treatments, it leads to increasing resistance. It is essential to use more potent and toxic antibiotics. Enzymes capable of hydrolyzing antibiotics are among the most common ways of resistance and TEM variants have been detected in several resistant isolates. Due to the rapid evolution of these variants, complex phenotypes have emerged and the need to understand their biological activity becomes crucial. To investigate the biochemical properties of TEM-180 and TEM-201 several computational methodologies have been used, allowing the comprehension of their structure and catalytic activity, which translates into their biological phenotype. In this work we intent to characterize the interface between these proteins and the several antibiotics used as ligands. We performed explicit solvent molecular dynamics (MD) simulations of these complexes and studied a variety of structural and energetic features. The interfacial residues show a distinct behavior when in complex with different antibiotics. Nevertheless, it was possible to identify some common Hot Spots among several complexes – Lys73, Tyr105 and Glu166. The structural changes that occur during the Molecular Dynamic (MD) simulation lead to the conclusion that these variants have an inherent capacity of adapting to the various antibiotics. This capability might be the reason why they can hydrolyze antibiotics that have not been described until now to be degraded by TEM variants. The results obtained with computational and experimental methodologies for the complex with Imipenem have shown that in order to this type of enzymes be able to acylate the antibiotics, they need to be capable to protect the ligand from water molecules.

Mixed extractant systems for metal recovery

Hoje em dia, a prevenção dos resíduos de metais é uma questão muito importante para um grande número de empresas, pois necessitam optimizar o seu sistema de tratamento de águas residuais a fim de alcançarem os limites legais dos teores em iões metálicos e poderem efectuar a descarga das águas residuais no domínio hídrico público. Devido a esta problemática foram efectuados estudos inovadores relacionados com a remoção de iões metálicos de águas residuais, verificando-se que as tecnologias de membrana oferecem uma série de vantagens para o efeito. Uma dessas tecnologias, referida como Membrana Líquida de Suporte (SLM), é baseada num mecanismo de extracção. A membrana hidrofóbica, impregnada com uma solução extractora, funciona como barreira entre a água residual e uma solução, geralmente ácida. A diferença de pH entre a água residual e a solução actua como força motriz para o transporte de iões metálicos da água residual para a referida solução. Poderá ocorrer um problema de falta de estabilidade, resultante da possível fuga da solução extractora para fora dos poros das membranas. Estudos anteriores mostraram que os ácidos alquilfosfóricos ou ácidos fosfónicos, como os reagentes D2EHPA e CYANEX e hidroxioximas como o LIX 860-I podem ser muito úteis para a extração de iões metálicos como ferro, cobre, níquel, zinco e outros. A clássica extracção líquido-líquido também tem mostrado que a mistura de diferentes extractores pode ter um efeito sinergético. No entanto, não é claro que haja um efeito óptimo da razão de extractor ou que tipo de complexo é formado durante o processo de extracção. O objectivo deste projecto é investigar este comportamento sinergético e as complexas formações por meio de um método espectrofotométrico, o “Job’s method” e “Mole-ratio method”. Estes métodos são utilizados para estimar a estequiometria dos vários complexos entre dois solutos, a partir da variação de absorvância dos complexos quando comparado com a absorvância do soluto. Com este projecto, o Job’s method e mole-ratio method serão aplicados a um sistema de três componentes, para conseguir mais informações sobre a complexação de níquel (II) e a fim de determinar a razão extractor: metal dos complexos formados durante a aplicação de mistura de extractores D2EHPA e LIX 860-I. Segundo Job’s method a elavada absorvância situa-se na região de 0,015-0,040 M de LIX 860-I e uma baixa concentração de D2EHPA. Quando as diferentes experiências são encontradas num conjunto experimental foram avaliadas de acordo com o método de trabalho, o valor máximo do gráfico foi encontrado para uma baixa fração molar do ião metálico e uma maior concentração de D2EHPA. Esta mudança foi encontrado de 0,50 até 0,30, que poderia apontar para a direção da formação de diferentes complexos. Para o Mole-Ratio method, a estequiometria dos complexos metal pode ser determinada a partir do ponto de intersecção das linhas tangente do gráfico da absorbância versus a concentração do ligante. Em todos os casos, o máximo foi obtido em torno de uma concentração total de 0,010 M. Quando D2EHPA foi aplicado sozinho, absorvâncias muito baixos foram obtidas.

Gold electrode modified by self-assembled monolayers of thiols to determine DNA sequences hybridization

The process of immobilization of biological molecules is one of the most important steps in the construction of a biosensor. In the case of DNA, the way it exposes its bases can result in electrochemical signals to acceptable levels. The use of self-assembled monolayer that allows a connection to the gold thiol group and DNA binding to an aldehydic ligand resulted in the possibility of determining DNA hybridization. Immobilized single strand of DNA (ssDNA) from calf thymus pre-formed from alkanethiol film was formed by incubating a solution of 2-aminoethanothiol (Cys) followed by glutaraldehyde (Glu). Cyclic voltammetry (CV) was used to characterize the self-assembled monolayer on the gold electrode and, also, to study the immobilization of ssDNA probe and hybridization with the complementary sequence (target ssDNA). The ssDNA probe presents a well-defined oxidation peak at +0.158 V. When the hybridization occurs, this peak disappears which confirms the efficacy of the annealing and the DNA double helix performing without the presence of electroactive indicators. The use of SAM resulted in a stable immobilization of the ssDNA probe, enabling the hybridization detection without labels. This study represents a promising approach for molecular biosensor with sensible and reproducible results.

Stability of the Transthyretin Molecule as a Key Factor in

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/ABeta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4- (3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

Optimizing potentiometric ionophore and electrode design for environmental on-site control of antibiotic drugs: Application to sulfamethoxazole

Potentiometric sensors are typically unable to carry out on-site monitoring of environmental drug contaminants because of their high limits of detection (LODs). Designing a novel ligand material for the target analyte and managing the composition of the internal reference solution have been the strategies employed here to produce for the first time a potentiometric-based direct reading method for an environmental drug contaminant. This concept has been applied to sulfamethoxazole (SMX), one of the many antibiotics used in aquaculture practices that may occur in environmental waters. The novel ligand has been produced by imprinting SMX on the surface of graphitic carbon nanostructures (CN) < 500 nm. The imprinted carbon nanostructures (ICN) were dispersed in plasticizer and entrapped in a PVC matrix that included (or not) a small amount of a lipophilic additive. The membrane composition was optimized on solid-contact electrodes, allowing near-Nernstian responses down to 5.2 μg/mL and detecting 1.6 μg/mL. The membranes offered good selectivity against most of the ionic compounds in environmental water. The best membrane cocktail was applied on the smaller end of a 1000 μL micropipette tip made of polypropylene. The tip was then filled with inner reference solution containing SMX and chlorate (as interfering compound). The corresponding concentrations were studied for 1 × 10−5 to 1 × 10−10 and 1 × 10−3 to 1 × 10−8 mol/L. The best condition allowed the detection of 5.92 ng/L (or 2.3 × 10−8 mol/L) SMX for a sub-Nernstian slope of −40.3 mV/decade from 5.0 × 10−8 to 2.4 × 10−5 mol/L.