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Master's Dissertation
DOI
https://doi.org/10.11606/D.76.2013.tde-20032013-091148
Document
Author
Full name
Vitor Hugo Balasco Serrão
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2013
Supervisor
Committee
Thiemann, Otavio Henrique (President)
Heel, Marin van
Lima, Luis Mauricio Trambaioli da Rocha e
Title in Portuguese
Complexos macromoleculares da via específica de incorporação de selênio de Escherichia coli
Keywords in Portuguese
Complexos macromoleculares
Interação proteína-proteína
Selenocisteína
Abstract in Portuguese
A existência de uma maior variedade de aminoácidos codificados pelo código genético tem estimado estudos sobre os mecanismos de síntese, reconhecimento e incorporação desses resíduos nas cadeias polipeptídicas nascentes. Um exemplo é a via de incorporação de selenocisteína evento cotraducional dirigido pelo códon UGA. Em bactérias, essa via conta com uma complexa maquinaria molecular composta por: Selenocisteína Sintase (SelA), Fator de Elongação Específico de Reconhecimento (SelB), Selenofosfato Sintetase (SelD), tRNA específico (SelC ou tRNAsec), sequência específica no mRNA (Sequência de Inserção de Selenocisteínas - SECIS) e Aminoacil tRNA Sintetase (aaRS). Pelo fato do selênio ter uma toxicidade elevada em ambientes celulares, é fundamental a compreensão do mecanismo catalítico e razão estequiométrica na formação dos complexos da via na etapa de incorporação junto ao tRNAsec, bem como sua caracterização estrutural foram os objetivos deste trabalho. A proteína SelA foi expressa e purificada para utilização em análises envolvendo microscopia de força atômica, microscopia eletrônica de transmissão com contraste negativo e em gelo vítreo foram realizadas nos complexos SelA e SelA-tRNAsec, visando obter um modelo estrutural e a razão estequiométrica dos complexos. A fim de compreender o mecanismo de passagem do selênio, ensaios de anisotropia de fluorescência e de microcalorimetria, corroborados pelas análises de troca de hidrogênio-deutério acoplado a espectrometria de massa e espectroscopia de infravermelho, elucidaram a formação e estequiometria do complexo ternário SelAtRNA sec-SelD. Tentativas de cristalização e análises cristalográficas também foram realizadas, no entanto, sem sucesso. Com os resultados obtidos foi possível propor que o reconhecimento de SelD e, consequentemente, a entrega do selenofosfato, seja uma etapa crucial da via de incorporação de selenocisteínas.
Title in English
Macromolecular assemblies of selenium incorporation specific pathway in Escherichia coli
Keywords in English
Macromolecular assemblies
Protein-protein interactions
Selenocysteine
Abstract in English
The existence of a greate variety of amino acids encoded by the genetic code has stimulated the study of the mechanisms of synthesis, recognition and incorporation of these residues in the nascent polypeptide chains. An example of genetic code expansion is the selenocysteine incorporation pathway an event cotraducional by the UGA codon. In bacteria, this pathway has a complex molecular machinery comprised: Selenocysteine Synthase (SelA), Specific Elongation Factor (SelB), Selenophosphate Synthetase (SelD), tRNA-specific (SelC or tRNAsec), Specific mRNA Sequence (SElenocysteine Insertion Sequence - SECIS) and Aminoacyl tRNA Synthetase (aaRS). Because selenium has high toxicity in cellular environments; it is essential for cell survival the association of this compound with proteins, in this case, selenoprotens and the associated proteins involved in the selenocysteine synthesis. Therfore the understanding of the catalytic mechanism, stoichiometric ratio, protein complex formation with the tRNAsec, and its structural characterization were the objectives of this work. The SelA protein was expressed and purified to used in analyzes involving atomic force microscopy, transmission electron microscopy with negative stain and in vitreous ice were performed in the complex SelA and SelA-tRNAsec in order to obtain a structural model of the complex and the stoichiometric ratio of its components. To study the selenium association with protein of the synthesis pathway, fluorescence anisotropy assays and isothermal titration calorimetry corroborated by the analysis hydrogen-deuterium exchange coupled to mass spectrometry and infrared spectroscopy were employed.Crystallization attempts were made and preliminary crystallographic analyzes were also performed, however, so far unsuccessfuly. The results obtained were possible to develop the hypothesis about the SelD recognition and, consenquently, the selenophosphate delivery, a crucial stage of the selenocysteine incorporation pathway.
 
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Publishing Date
2013-03-21
 
WARNING: The material described below relates to works resulting from this thesis or dissertation. The contents of these works are the author's responsibility.
  • MANZINE, LIVIA REGINA, et al. Assembly stoichiometry of bacterial selenocysteine synthase and SelC (tRNAsec) [doi:10.1016/j.febslet.2013.02.014]. FEBS Letters [online], 2013, vol. 587, p. 1-5.
  • Serrão, Vitor Hugo Balasco, et al. Promiscuous interactions of human septins: The GTP binding domain of SEPT7 forms filaments within the crystal [doi:10.1016/j.febslet.2011.10.043]. FEBS Letters [online], 2011, vol. 585, p. 3868-3873.
  • ALESSANDRO, F, et al. Structure-function analysis of human septin 7 and 9. GTPase domain. In XXXVIII Annual Meeting of the Brazilian Biochemistry and Molecular Biology Society, Águas de Lindóia, 2009. Program and Index.São Paulo : Sociedade Brasileira de Bioquímica e Biologia Molecular, 2009. Resumo.
  • SERRAO, V. H., et al. Crystal Structures of Human Septins: Unveiling the Mechanism of Septin Bundling. In Brazilian Biochemistry and Molecular Biology Society (SBBq), Foz do Iguaçu/PA, 2011. XL Annual Meeting of SBBq., 2011. Resumo.
  • SERRAO, V. H., et al. Structural and biochemical studies of human septin 7. In XXXVII Reunião Anual da Sociedade Brasileira de Bioquímica e Biologia Molecular - SBBq, Águas de Lindóia, 2008. Program and Index., 2008. Resumo.
  • Thiemann, Otavio H., et al. Structural and biochemical studies of human septin 7. In 3rd International Workshop on Spectroscopy for Biology, Maresias, 2010. 3rd International Workshop on Spectroscopy for Biology., 2010. Resumo.
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