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CaracterizaĆ§Ć£o de um novo citocromo do tipo C que forma "nanofios" de Hemos em Geobacter Sulfurreducens
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Resumo(s)
O citocromo dodeca-hemico GSU1996, da bacteria Geobacter sulfurreducens, e uma
proteĆna de estrutura linear, constituĆda por quatro domĆnios (A a D) e que actua, putativamente, como ānanofioā molecular, transferindo eletrƵes entre proteĆnas periplasmĆ”ticas ou para proteĆnas da membrana externa.
Neste trabalho, testou-se a aplicaĆ§Ć£o de uma estratĆ©gia modular ao estudo deste novo e complexo citocromo c. Foi realizada a expressĆ£o heterĆ³loga em E. coli de cada domĆnio individual e do fragmento CD que constitui o GSU1996. Dado o baixo rendimento na expressĆ£o dos domĆnios A e B, testaram-se alguns factores com vista a optimizacao das suas condiƧƵes de expressĆ£o. No caso do domĆnio A, o melhor resultado ocorreu em E. coli JCB7123 induzida com 50 Ī¼M de IPTG, obtendo-se um rendimento final de 0,15 mg de proteĆna por litro de cultura.
Efectuou-se a atribuiĆ§Ć£o dos sinais aos substituintes hemicos dos domĆnios A, C e D no
estado reduzido atravĆ©s de experiencias de RessonĆ¢ncia MagnĆ©tica Nuclear. A abordagem modular foi validada na atribuiĆ§Ć£o de sinais ao fragmento CD, em que se utilizou como guia a atribuiĆ§Ć£o realizada nos domĆnios individuais que o compƵem.
Para determinaĆ§Ć£o de parĆ¢metros termodinĆ¢micos, realizaram-se testes de reoxidaĆ§Ć£o aos domĆnios A e D a pH 8,0 e 16Ā°C, tendo sido possĆvel identificar os perfis de oxidaĆ§Ć£o dos diferentes grupos hemo apenas no primeiro caso. Contrariamente ao esperado para um grupo hemo com coordenaĆ§Ć£o axial His-Met e analogamente ao observado no domĆnio C, o grupo hemo IV e o que sofre maior grau de oxidaĆ§Ć£o no primeiro passo de oxidaĆ§Ć£o-reduĆ§Ć£o, seguindo-se o grupo hemo I e, finalmente, o grupo hemo III.
TitulaƧƵes de oxidaĆ§Ć£o-reduĆ§Ć£o do domĆnio A seguidas por espectroscopia de UV-visĆvel indicam que nas condiƧƵes experimentais utilizadas existe reversibilidade entre os processos. O valor de Eapp e -130 mV, sendo muito semelhante ao do domĆnio D.
The dodecahemic cytochrome GSU1996, from the bacterium Geobacter sulfurreducens, is a linear structure protein, composed of four domains (A to D) which acts, putatively, as a molecular ānanowireā and transfers electrons between periplasmic or to outer membrane proteins. In the present work, the application of a modular strategy to the study of this new and complex cytochrome c was tested. Heterologous expression of each individual domain and tandem CD that constitute GSU1996 was performed in E. coli. Optimization was made in order to overcome the low yield obtained with domains A and B. Regarding domain A, the best result was attained through expression in E. coli JCB7123 induced with 50 Ī¼M IPTG, and a final yield of 0,15 mg of protein per liter of culture was obtained. The assignment of the signals from the heme substituents of domains A, C and D in the reduced state was accomplished through the spectra acquired with Nuclear Magnetic Resonance experiments. The modular approach was validated with the assignment of the signals to the tandem CD, in which was used the assignment of the individual domains C and D as a guide to achieve the tandemās assignment. In order to determine thermodynamic parameters, re-oxidation assays were performed with domains A and D. It was possible to identify the redox patterns of the different hemes at pH 8,0 and 16Ā°C only for domain A. Against expectations and analogously to domain C, heme IV, with axial coordination His-Met, was the most oxidized in the first oxidation step, followed by heme I and finally, heme III. Redox titrations from domain A monitored by UV-visible spectroscopy show that in the experimental conditions used there is reversibility between both processes. Eapp in these conditions is -130 mV and it is very similar to that of domain D.
The dodecahemic cytochrome GSU1996, from the bacterium Geobacter sulfurreducens, is a linear structure protein, composed of four domains (A to D) which acts, putatively, as a molecular ānanowireā and transfers electrons between periplasmic or to outer membrane proteins. In the present work, the application of a modular strategy to the study of this new and complex cytochrome c was tested. Heterologous expression of each individual domain and tandem CD that constitute GSU1996 was performed in E. coli. Optimization was made in order to overcome the low yield obtained with domains A and B. Regarding domain A, the best result was attained through expression in E. coli JCB7123 induced with 50 Ī¼M IPTG, and a final yield of 0,15 mg of protein per liter of culture was obtained. The assignment of the signals from the heme substituents of domains A, C and D in the reduced state was accomplished through the spectra acquired with Nuclear Magnetic Resonance experiments. The modular approach was validated with the assignment of the signals to the tandem CD, in which was used the assignment of the individual domains C and D as a guide to achieve the tandemās assignment. In order to determine thermodynamic parameters, re-oxidation assays were performed with domains A and D. It was possible to identify the redox patterns of the different hemes at pH 8,0 and 16Ā°C only for domain A. Against expectations and analogously to domain C, heme IV, with axial coordination His-Met, was the most oxidized in the first oxidation step, followed by heme I and finally, heme III. Redox titrations from domain A monitored by UV-visible spectroscopy show that in the experimental conditions used there is reversibility between both processes. Eapp in these conditions is -130 mV and it is very similar to that of domain D.
DescriĆ§Ć£o
Tese de mestrado em BioquĆmica, apresentada Ć Universidade de Lisboa, atravĆ©s da Faculdade de CiĆŖncias, 2012
Palavras-chave
Citocromos C multi-hĆ©micos "Nanofios" de hemos Geobacter sulfurreducens ExpressĆ£o heterĆ³loga RessonĆ¢ncia magnĆ©tica nuclear Teses de mestrado - 2012