Crystallographic studies on membrane and cytoplasmic enzymes

Dissertation presented to obtain the Ph.D degree in Biochemistry, Structural Biochemistry

Characterization of the [NiFe] Hydrogenase from the sulfate reducer Desulfovibrio vulgaris Hildenborough

The [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough was isolated from the cytoplasmic membranes and characterized by EPR spectroscopy. It has a total molecular mass of 98.7 kDa (subunits of 66.4 and 32.3 kDa), and contains 1 nickel and 12 Fe atoms per heterodimer. The catalytic activities for hydrogen consumption and production were determined to be 174 and 89 umol H2 min-1 mg -1, respectively. As isolated, under aerobic conditions, this hydrogenase exhibits EPR signals characteristic of the nickel centers in [NiFe] hydrogenases (Ni-A signal at gx,y,z=2.32, 2.23 and ~2.0 and Ni-B signal at gx,y,z=2.33, 2.16 and ~2.0) as well as an intense quasi-isotropic signal centered at g=2.02 due to the oxidized [3Fe-4S] center. The redox profile under hydrogen atmosphere is remarkably similar to that of other [NiFe] hydrogenases. The signals observed for the oxidized state disappear, first being substituted by the Ni-C type signal (gx,y,z=2.19, 2.14, ~2.01), which upon long incubation under hydrogen yields the split Ni-C signal due to interaction with the reduced [4Fe-4S] centers.

Mitosis and protein Na-terminal acetylation

Dissertation presented to obtain the Ph.D degree in Developmental Biology

Análise da estrutura e da função da proteína morfogenética RodZ de Bacillus subtilis

Resumo: RodZ é um componente do sistema morfogenético das células bacterianas. É uma proteína transmembranar que localiza em bandas ao longo do eixo longitudinal da célula. Em Bacillus subtilis, RodZ consiste numa porção citoplasmática, RodZn, e em uma parte extra-citoplasmática, RodZc. RodZn contém um domínio em helixturn- helix (HTH), enquanto que RodZc pode ser dividido num domínio coiled-coil e num domínio terminal C, de função desconhecida. Um segmento transmembranar (TM) único separa RodZn de RodZc. A eliminação de rodZ causa alongamento do nucleóide e leva à produção de células polares nucleadas. Aqui, mostramos que RodZn é estruturado, estável e em hélice α. Descobrimos que as substituições Y32A e L33A na suposta hélice de reconhecimento (3) do motivo HTH, bem como as substituições Y49A e F53A, fora do motivo HTH (4), causam divisão assimétrica, mas apenas as últimas levam à deslocalização sub-celular de RodZ. Sugerimos que as hélices 3 e 4 são utilizadas para uma interacção proteína-proteína ou proteína- DNA essencial para divisão celular enquanto que 4 deve contactar um componente do citosqueleto, possivelmente MreB, uma vez que a correcta localização sub-celular de RodZ depende desta proteína. Em todos os mutantes as células polares são anucleadas, pelo que concluímos que o alongamento do nucleóide não é um prérequisito para divisão assimétrica. RodZc é largamente não estruturado mas com conteúdo de folha , sendo estabilizado pelo domínio coiled-coil. Mostramos uma relação homóloga entre RodZc e a bomba de transporte Na+/Ca2+ NCX1 e identificámos dois resíduos no domínio C, G265 e N275, essenciais para a manutenção da forma celular. Estes resíduos fazem parte de um motivo em gancho que pode actuar como um local de interacção com um ligando desconhecido. RodZn e RodZc são monoméricos em solução. Contudo, na membrana, RodZ interage consigo própria num sistema de dois híbridos (Split-Ubiquitin) em levedura, sugerindo que possa formar multímeros in vivo.-----------ABSTRACT: RodZ is a transmembrane component of the bacterial core morphogenic apparatus. RodZ localizes in bands long the longitudinal axis of the cell, and it is though to functionally link the cell wall to the actin cytoskeleton. In Bacillus subtilis, RodZ consists of a cytoplasmic moiety, RodZn, and an extracytoplasmic moiety, RodZc. RodZn contains a predicted helix-turn-helix domain, whereas RodZc is thought to contain a coiled-coil region and a terminal C domain of unknown function. A single transmembrane domain separates RodZn from RodZc. Deletion of rodZ causes elongation of the nucleoid and leads to the production of polar minicells containing DNA. Here, we have studied the structure and function of RodZn and RodZc. We show that RodZn is a stable, folded, -helical domain. We discovered that the Y32A and L33A substitutions within the presumptive recognition helix (3) of the HTH motif, as well as the Y49A and F53A substitutions outside of the HTH motif (in 4) cause asymmetric cell division. However, only the substitutions in 4 cause sub-celular delocalization of RodZ. We suggest that 3 and 4 are used for a protein-protein or protein-DNA interaction important for cell division, whereas 4 is likely to contact a cytoskeletal component, presumably MreB. The polar cells formed by all the mutants are anucleate. We conclude that nucleoid elongation is not a prerequisite for asymmetric division. RodZc appears to be a largely unstructured domain, with some -sheet content, and is stabilized by the coiled-coil region. We show a homology relationship between RodZc and the NCX1 Na+/Ca2+ transporter and we found two residues within the C domain, G265 and N275, that are important for cell shape determination. These residues are predicted to be essential determinants of a claw-like motif, which may act as a binding site for an unknown ligand. Both the isolated RodZn and RodZc proteins are monomeric in solution. However, because full-length RodZ interacts with itself in a split-ubiquitin yeast two-hybrid assay, we suggest that it may dimerize or form higher order multimers in vivo.

Analysis of the intranuclear life of nonsense transcripts

Dissertação para obtenção do Grau de Doutor em Biologia, Especialidade de Biologia Molecular

Dissecting cross-talk between microglia and motoneurons in ALS: signaling events and soluble factors

Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina

Using cilia mutants to study left-right asymmetry in zebrafish

A thesis submitted in fulfillment of the requirements for the degree of the Masters in Molecular Genetics and Biomedicine

Deciphering the genome of Desulfovibrio gigas: the role of the hydrogenases

The work presented in this thesis describes the functional characterization of hydrogenases in the overall energy metabolism of the sulfate reducing bacterium Desulfovibrio gigas. With the complete annotation of the D. gigas genome, we were able to verify that only the two previously described hydrogenases are present in this organism, the periplasmic [NiFe] HynAB and the cytoplasmic membrane-bound [NiFe] Ech.(...)

Genomic and environmental factors influence Wolbachia-Drosophila symbiosis

Intracellular, vertically transmitted bacteria form complex and intimate relationships with their hosts. Wolbachia, maternally transmitted α- proteobacteria, live within the cells of numerous arthropod species. Wolbachia are famous master manipulators of insect reproduction: to favour their own spread they can induce male killing, parthenogenesis or cytoplasmic incompatibility. Wolbachia can also protect various insects from pathogens, which makes them a promising tool for the control of vector-borne diseases. Mosquitoes with Wolbachia have already been released in the wild to eliminate dengue. Yet, how Wolbachia manipulate their hosts remains largely unknown.(...)

Synthesis of precursors of the rare 3-O-methylmannose polysaccharides present in Nontuberculous Mycobacteria

3-O-methylmannose polysaccharides (MMPs) are cytoplasmic carbohydrates synthesized by mycobacteria, which play important intracellular roles, such as for example in metabolism regulation. An important way to confirm if the inhibition of the synthesis of these polysaccharides will critically affect the survival of mycobacteria is the study of the biosynthetic pathways from these molecules on these microorganisms. The purpose of this work is the efficient synthesis of three saccharides, which are rare cellular precursors from the biosynthesis of the mycobacterial polysaccharides, allowing its study. In order to obtain these molecules, a chemical strategy to connect two precursors was used. This process is called chemical glycosylation and its importance will be highlighted as an important alternative to enzymatic glycosylation. The first objective was the synthesis of the disaccharides Methyl (3-O-methyl-α-D-mannopyranosyl)-(1→4)-3-O-methyl-α-D-mannopyranoside and (3-O-Methyl-α-D-mannopyra- nosyl)-(1→4)-3-O-methyl-(α/β)-D-mannopyranose. The mannose precursors were prepared before the glycosylation reaction. The same mannosyl donor was used in the preparation of both molecules and its efficient synthesis was achieved using a 8 step synthetic route from D-mannose. A different mannosyl acceptor was used in the synthesis of each disaccharide and their syntheses were also efficient, the first one a 4 step synthetic route from α-methyl-D-mannose and the second one as an intermediate from the synthesis of the mannosyl donor. The stereoselective preparation of these disaccharides was performed successfully. The second and last objective of the proposed work was the synthesis of the tetrasaccharide methyl (3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyra- nosyl-(1→4)-3-O-methyl-α-D-mannopyranosyl-(1→4)-3-O-methyl-α-D-mannopyranoside. The disaccharide acceptor and donor to be linked through a stereoselective glycosidic reaction had to be first synthesized. Several synthetic strategies were studied. Neither the precursors nor the tetrasaccharide were synthesized, but a final promising synthetic route for its preparation has been proposed.