Caracterização da interação HipAB na Shewanella oneidensis MR1

• Autor(a) principal: Henriques, Andreia Filipa Pereira
• Data de Publicação: 2013
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/11775
• topic: Bioquímica - Teses de mestrado; Bactérias patogénicas; Resistência a antibióticos; Toxinas; Proteínas

Bacterial resistance is a major issue in research related to environmental and public health, due to the ineffectiveness of antibiotics, resulting from a subpopulation of bacterial cells entering a dormant state. Multiple studies revealed that toxin-antitoxin systems are involved in such bacterial persistence. An example of such a toxin-antitoxin system is the HipAB complex, where HipA, and its cognate antitoxin are intimately related. HipB is very unstable and it is prone to degradation by proteases (e.g. Lon protease), leaving HipA exerting its toxic effect on cells. To study the conformations and interactions from HipAB in Shewanella oneidensis, it was necessary to overexpress and purify the proteins and to perform cross linking reactions which were analysed by mass spectrometry. The cross linking reactions were performed with BS3 (Bis[sulfosuccinimidyl] suberate), an in solution cross linker, tested at different concentrations (0.5mM, 1mM, 2mM) to find more efficient reaction conditions. By SDS-PAGE analysis it was possible to conclude that the best cross linker concentration is the highest tested. Furthermore, some predictions from possible intra- and intermolecular binding sites were made, unfortunately it was only possible to identify an interaction between HipA(143) and HipA(264).