A further investigation of the cytochrome b5–cytochrome c complex

J Biol Inorg Chem (2003) 8: 777–786 DOI 10.1007/s00775-003-0479-y

A further investigation of the cytochrome b5–cytochrome c complex

J Biol Inorg Chem (2003) 8: 777–786

Diversidade genética do vírus da Hepatite C circulante numa população de utilizadores de drogas injectáveis de Lisboa

Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e Tecnologia

Dynamic and interaction of cytochrome c with Pf1 virus

Dissertação apresentada na Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa para obtenção do grau de Mestre em BioOrgânica

Gd(III) chelates as NMR probes of protein-protein interactions. Case study: rubredoxin and cytochrome c3

Inorganic Chemistry 50(21):10600-7

An NMR structural study of nickel-substituted rubredoxin

J Biol Inorg Chem (2010) 15:409–420 DOI 10.1007/s00775-009-0613-6

Benefits of membrane electrodes in the electrochemistry of metalloproteins: mediated catalysis of Paracoccus pantotrophus cytochrome c peroxidase by horse cytochrome c: a case study

J Biol Inorg Chem. 2008 Jun;13(5):779-87. doi: 10.1007/s00775-008-0365-8

A copper protein and a cytochrome bind at the same site on bacterial cytochrome c peroxidase

Biochemistry, 2004, 43 (46), pp 14566–14576 DOI: 10.1021/bi0485833

Molecular determinants of ligand specificity in carbohydrate-binding modules: an NMR and X-ray crystallography integrated study

Dissertação para obtenção do Grau de Doutor em Bioquímica – Ramo Bioquímica Estrutural

Controlo de reacções de polimerização em miniemulsão através de técnicas de espectroscopia NIR e Raman

Dissertação para obtenção do Grau de Mestre em Engenharia Química e Bioquímica

Application of NMR RDC’s, relaxation and self-diffusion for the study of dynamic processes of small molecules in solution

Spin-lattice Relaxation, self-Diffusion coefficients and Residual Dipolar Couplings (RDC’s) are the basis of well established Nuclear Magnetic Resonance techniques for the physicochemical study of small molecules (typically organic compounds and natural products with MW < 1000 Da), as they proved to be a powerful and complementary source of information about structural dynamic processes in solution. The work developed in this thesis consists in the application of the earlier-mentioned NMR techniques to explore, analyze and systematize patterns of the molecular dynamic behavior of selected small molecules in particular experimental conditions. Two systems were chosen to investigate molecular dynamic behavior by these techniques: the dynamics of ion-pair formation and ion interaction in ionic liquids (IL) and the dynamics of molecular reorientation when molecules are placed in oriented phases (alignment media). The application of NMR spin-lattice relaxation and self-diffusion measurements was applied to study the rotational and translational molecular dynamics of the IL: 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4]. The study of the cation-anion dynamics in neat and IL-water mixtures was systematically investigated by a combination of multinuclear NMR relaxation techniques with diffusion data (using by H1, C13 and F19 NMR spectroscopy). Spin-lattice relaxation time (T1), self-diffusion coefficients and nuclear Overhauser effect experiments were combined to determine the conditions that favor the formation of long lived [BMIM][BF4] ion-pairs in water. For this purpose and using the self-diffusion coefficients of cation and anion as a probe, different IL-water compositions were screened (from neat IL to infinite dilution) to find the conditions where both cation and anion present equal diffusion coefficients (8% water fraction at 25 ºC). This condition as well as the neat IL and the infinite dilution were then further studied by 13C NMR relaxation in order to determine correlation times (c) for the molecular reorientational motion using a mathematical iterative procedure and experimental data obtained in a temperature range between 273 and 353 K. The behavior of self-diffusion and relaxation data obtained in our experiments point at the combining parameters of molar fraction 8 % and temperature 298 K as the most favorable condition for the formation of long lived ion-pairs. When molecules are subjected to soft anisotropic motion by being placed in some special media, Residual Dipolar Couplings (RDCs), can be measured, because of the partial alignment induced by this media. RDCs are emerging as a powerful routine tool employed in conformational analysis, as it complements and even outperforms the approaches based on the classical NMR NOE or J3 couplings. In this work, three different alignment media have been characterized and evaluated in terms of integrity using 2H and 1H 1D-NMR spectroscopy, namely the stretched and compressed gel PMMA, and the lyotropic liquid crystals CpCl/n-hexanol/brine and cromolyn/water. The influence that different media and degrees of alignment have on the dynamic properties of several molecules was explored. Different sized sugars were used and their self-diffusion was determined as well as conformation features using RDCs. The results obtained indicate that no influence is felt by the small molecules diffusion and conformational features studied within the alignment degree range studied, which was the 3, 5 and 6 % CpCl/n-hexanol/brine for diffusion, and 5 and 7.5 % CpCl/n-hexanol/brine for conformation. It was also possible to determine that the small molecules diffusion verified in the alignment media presented close values to the ones observed in water, reinforcing the idea of no conditioning of molecular properties in such media.

Characterization of novel heme-containing sensor proteins from Geobacter sulfurreducens

The focus of this Thesis was the study of the sensor domains of two heme-containing methyl-accepting chemotaxis proteins (MCP) from Geobacter sulfurreducens: GSU0582 and GSU0935. These domains contain one c-type heme, form swapped dimers with a PAS-like fold and are the first examples of a new class of heme sensors. NMR spectroscopy was used to assign the heme and polypeptide signals in both sensors, as a first step to probe conformational changes in the vicinity of the hemes. However, the presence of two conformations in solution impaired the confident assignment of the polypeptide signals. To understand how conformational changes and swapped dimerization mechanism can effectively modulate the function of the two sensor domains and their signal transduction process, the sensor domains folding and stability were studied by circular dichroism and UV-visible spectroscopy. The results showed differences in the thermodynamic stability of the sensors, with GSU0582 displaying higher structural stability. These studies also demonstrated that the heme moiety undergoes conformational changes matching those occurring at the global protein structure and that the content of intrinsically disordered segments within these proteins (25% for GSU0935; 13% for GSU0582) correlates with the stability differences observed. The thermodynamic and kinetic properties of the sensor domains were determined at different pH and ionic strength by visible spectroscopy and stopped-flow techniques. Despite the remarkably similar spectroscopic and structural features of the two sensor domains, the results showed that their properties are quite distinct. Sensor domain GSU0935 displayed more negative reduction potentials and smaller reduction rate constants, which were more affected by pH and ionic strength. The available structures were used to rationalize these differences. Overall, the results described in this Thesis indicate that the two G. sulfurreducens MCP sensor domains are designed to function in different working potential ranges, allowing this bacterium to trigger an adequate cellular response in distinct anoxic subsurface environments.

Espectroscopia ótica de emissão em descargas luminescentes anómalas para produção de filmes finos

O objetivo deste projeto é implementar e explorar um sistema de espectroscopia ótica de emissão para monitorizar e tentar correlacionar os espectros óticos de emissão da descarga produzida por mais que um cátodo com a composição de filmes finos com gradiente em profundidade obtidos por co-deposição utilizando dois cátodos magnetrão. O projeto foi desenvolvido Laboratório de Plasmas e Aplicações da Linha 2/CEFITEC. Numa primeira fase reativou-se o sistema de deposição de filmes binários (SIDEBI) e um dos cátodos magnetrão, para funcionar no modo de obtenção de filmes em codeposição com dois cátodos. O sistema foi reconfigurado, testado, detetando e corrigindo anomalias de funcionamento. Na segunda fase do projeto instalou-se o acoplamento da fibra ótica a câmara de vácuo. Para a aquisição dos espectros, adaptou-se um programa em LabView 13.0 de controlo simultâneo das fontes de tensão já existente, para que este possa controlar o espectrómetro ótico AvaSpec-3648-USB2 da Avantes e adquirir os espectros óticos de emissão da descarga obtida pelos cátodos. Na fase final dos trabalhos produziram-se filmes com gradiente de composição em profundidade e analisaram-se os espectros óticos obtidos em cada etapa da deposição para definir as linhas espectrais a serem monitorizadas e a sua relação com as taxas de deposição estimadas de cada metal. A composição dos filmes foi posteriormente analisada por RBS e os seus resultados comparados com os estimados a partir das curvas de calibração da potência da descarga em função das taxas de deposição dos materiais. O capítulo 1 apresenta uma introdução aos temas das descargas luminescentes anómalas, deposição catódica e espectroscopia ótica de emissão. No capítulo 2 são apresentados os diversos sistemas utilizados na realização do trabalho experimental. Numa primeira fase apresenta-se o sistema de deposição de filmes por pulverização catódica. Numa segunda fase são descritas as modificações efetuadas a este sistema de deposição necessárias para a implementação de um sistema de espectroscopia ótica de emissão. É também aqui descrito método de implementação deste sistema de espectroscopia e a sua constituição. O capítulo 3 descreve o método abordado para tratamento dos espectros de emissão obtidos nas descargas, e são apresentados os resultados obtidos e a respetiva análise. No capítulo 4 são apresentadas as conclusões referentes ao trabalho realizado.