Morgado, L, Fernandes AP, Dantas JM, Silva MA, Salgueiro CA.
2012.
On the road to improve the bioremediation and electricity-harvesting skills of Geobacter sulfurreducens: functional and structural characterization of multihaem cytochromes. Biochemical Society transactions. 40(6):1295-1301.
AbstractExtracellular electron transfer is one of the physiological hallmarks of Geobacter sulfurreducens, allowing these bacteria to reduce toxic and/or radioactive metals and grow on electrode surfaces. Aiming to functionally optimize the respiratory electron-transfer chains, such properties can be explored through genetically engineered strains. Geobacter species comprise a large number of different multihaem c-type cytochromes involved in the extracellular electron-transfer pathways. The functional characterization of multihaem proteins is particularly complex because of the coexistence of several microstates in solution, connecting the fully reduced and oxidized states. NMR spectroscopy has been used to monitor the stepwise oxidation of each individual haem and thus to obtain information on each microstate. For the structural study of these proteins, a cost-effective isotopic labelling of the protein polypeptide chains was combined with the comparative analysis of 1H-13C HSQC (heteronuclear single-quantum correlation) NMR spectra obtained for labelled and unlabelled samples. These new methodological approaches allowed us to study G. sulfurreducens haem proteins functionally and structurally, revealing functional mechanisms and key residues involved in their electron-transfer capabilities. Such advances can now be applied to the design of engineered haem proteins to improve the bioremediation and electricity-harvesting skills of G. sulfurreducens.
Dantas, JM, Morgado L, Londer YY, Fernandes AP, Louro RO, Pokkuluri PR, Schiffer M, Salgueiro CA.
2012.
Pivotal role of the strictly conserved aromatic residue F15 in the cytochrome c7 family. Journal of Biological Inorganic Chemistry. 17(1):11-24.
AbstractCytochromes c7 are periplasmic triheme proteins that have been reported exclusively in δ-proteobacteria. The structures of five triheme cytochromes identified in Geobacter sulfurreducens and one in Desulfuromonas acetoxidans have been determined. In addition to the hemes and axial histidines, a single aromatic residue is conserved in all these proteins - phenylalanine 15 (F15). PpcA is a member of the G. sulfurreducens cytochrome c7 family that performs electron/proton energy transduction in addition to electron transfer that leads to the reduction of extracellular electron acceptors. For the first time we probed the role of the F15 residue in the PpcA functional mechanism, by replacing this residue with the aliphatic leucine by site-directed mutagenesis. The analysis of NMR spectra of both oxidized and reduced forms showed that the heme core and the overall fold of the mutated protein were not affected. However, the analysis of 1H-15N heteronuclear single quantum coherence NMR spectra evidenced local rearrangements in the α-helix placed between hemes I and III that lead to structural readjustments in the orientation of heme axial ligands. The detailed thermodynamic characterization of F15L mutant revealed that the reduction potentials are more negative and the redox-Bohr effect is decreased. The redox potential of heme III is most affected. It is of interest that the mutation in F15, located between hemes I and III in PpcA, changes the characteristics of the two hemes differently. Altogether, these modifications disrupt the balance of the global network of cooperativities, preventing the F15L mutant protein from performing a concerted electron/proton transfer.
Morgado, L, Paixão VB, Schiffer M, Pokkuluri PR, Bruix M, Salgueiro CA.
2012.
Revealing the structural origin of the redox-Bohr effect: the first solution structure of a cytochrome from Geobacter sulfurreducens. Biochemical Journal. 441(1):179-187.
AbstractGs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.
Maia, MJ, Moiteiro AI, Horstink L, Farelo M, Antunes R.
2012.
{Análise de um processo decisório controverso: a co-incineração em Souselas [Analysis of a controversial decision process: the co-incineration at Souselas]}. , Number 10/2012: Universidade Nova de Lisboa, IET/CICS.NOVA-Interdisciplinary Centre on Social Sciences, Faculty of Science and Technology
AbstractThe scientific controversy generated around the destiny given to the fraction of hazardous industrial waste produced in Portugal and how the country dealt with this situation was the stand out point in Souselas case. Here, the dominant aspect of the analysis focused on the implementation of a solution for the treatment of hazardous industrial waste. These wastes result from industrial processes contain or are contaminated, by substances that, at certain concentrations, represent a risk to human health or to the environment. Their treatment can be done using co-incineration in existing cement factories. Having in mind the environment analysis of a controversial process, through the statements made by the different actors involved, the case of Souselas was our object of study. Initially, the actors involved in the process were identified and characterized, in terms of position, interests and / or concerns. This analysis has strengthened with the gathering of documentary elements of analysis. In a second phase the historical process was prepared. Only then, the conditions to make an interpretation of what really happened in the process were gathered, then , it was possible to identify which parts were successful and unsuccessful, and to interpret “why” these successes and failures occurred. Thus, after the identification of key variables and leverage points, a causal diagram and a schematic simulation of the behaviour of reference in case Souselas was designed. We conclude that the process of Souselas was a significant milestone with regard to social organization and spontaneous local actors in situations of opposition to central government decisions with local impact. It was also a turning point in governance according to the model of representative democracy, whose technocratic and elitist character is called into question. The Souselas case emphasized itself as a microcosm on the conflict of interests that we find at a global level heightened since the 90s and that
Sequeira, S{\'ılvia, Cabrita EJ, Macedo FM.
2012.
Antifungals on paper conservation: An overview. International Biodeterioration & Biodegradation. 74:67-84.
AbstractSince its invention, paper has become one of the main carriers of our cultural, scientific, political, economic and historical information. Given the importance of this material, its preservation is a matter of great interest. Paper can be deteriorated due to physical, chemical and biological agents. Within microorganisms, fungi are the major paper biodeteriogens. Throughout history, several methods have been used to prevent and stop fungal deterioration on paper based materials. In this work we present a review of the main chemical and physical methods used to avoid fungal paper biodeterioration until nowadays and also of some new approaches tested recently. The advantages and disadvantages of these methods are discussed as well as their health effects. Studies regarding antifungal compositions, methods of application, performance and effects on the treated materials are also presented with the aim of providing a clear set of conclusions on the topic. (C) 2012 Elsevier Ltd. All rights reserved.
Ferraz, R, Branco LC, Marrucho IM, Araujo JMM, Rebelo LPN, da Ponte MN, Prudencio C, Noronha JP, Petrovski Z.
2012.
Development of novel ionic liquids based on ampicillin. Medchemcomm. 3:494-497., Number 4
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Coelho, C, Mahro M, Trincao J, Carvalho ATP, Ramos MJ, Terao M, Garattini E, Leimkuehler S, Romao MJ.
2012.
The First Mammalian Aldehyde Oxidase Crystal Structure INSIGHTS INTO SUBSTRATE SPECIFICITY. Journal of Biological Chemistry. 287:40690-40702., Number 48
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Kowacz, M, Mukhopadhyay A, Carvalho AL, Esperanca J, Romao MJ, Rebelo LPN.
2012.
Hofmeister effects of ionic liquids in protein crystallization: Direct and water-mediated interactions. Crystengcomm. 14:4912-4921., Number 15
AbstractWe have performed experiments on the crystallization of two low molecular weight, positively charged proteins, lysozyme and ribonuclease A, using ionic liquids as either crystallization additives or, in particular cases, as precipitating agents. The ionic liquids (ILs) have been ordered according to their salting-in/out ability and the relative position of these ionic liquids in this ranking has been rationalized by considering their hydration properties (positive-negative, hydrophobic-hydrophilic). The ability to screen the effective charge of cationic proteins and aid protein nucleation (salting-out) has been shown to be superior for large polarizable anions with low charge density, negatively hydrated-Cl-, Br-, [SCN](-), methane-[C1SO3](-) and ethanesulfonates [C2SO3](-), than for anions with a relatively stable hydration shell, positively hydrated-lactate [Lac](-), butylsulfonate [C4SO3](-) and acetate [Ac](-). Upon increasing the background salt concentration, where electrostatic interactions are already effectively screened, the ability of the IL ions to stabilize proteins in solution (salting-in) has been shown to increase as the ions are likely to migrate to the non-polar protein surface and lower protein-water interfacial tension. This tendency is enhanced as the focus moves from those ions with positively hydrated hydrophilic compartments (e. g. [Ac](-)) to those with negatively hydrated groups (e. g. [C1SO3](-)) and the prevailing hydrophobic hydration (e. g. [C4SO3](-)). The observed inversion in the relative effect of ILs on protein crystallization with increasing ionic strength of the aqueous media has been interpreted as the differing effects of ion adsorption: charge screening and interfacial tension modification. Moreover, this work can further help in our understanding of the influence of ionic liquids on conformational changes of biomacromolecules in solution. Identification of the specific incorporation sites for choline and acetate ions, localized in two lysozyme crystals grown in pure IL solutions without any buffer or inorganic precipitant, can give us some insight into the role of the ionic liquid ions in protein structure development.