Caracterização Estrutural Da Proteína Csm2, Uma Proteína Do Sistema Crispr-Cas

• Ano de defesa: 2018
• Autor(a) principal: Enamorado, Gloria Alejandra Gallo [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Strutural Caracterization; Protein Csm2; Protein Do Sistem Crispr-Cas; Caracterização Estrutural; Proteína Csm2; Proteína; Sistema Crispr-Cas
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=6548061; https://repositorio.unifesp.br/handle/11600/52834

The clusters of regularly interspaced short palindromic repeats (CRISPR) and the Cas (CRISPR‐associated) proteins form an adaptive immune system in bacteria and archaea that evolved as an RNA‐guided interference mechanism to target and degrade foreign genetic elements. This system consists of an operon composed of regions of identical repeats, separated by variable spacers derived from invading nucleic acids. Together with the Cas proteins, this system forms an adaptive and heritable immune system. The main objective of this project was to clone, recombinantly express and purify the Csm2 protein from Thermotoga maritima MSB8, for biochemical and structural characterization. This protein is part of a marginally studied complex of Cas ribonucleoproteins (RNP) termed the Csm complex, which is involved in the targeting of exogenous DNA. The protein was recombinantly produced in Escherichia coli BL21(DE3) and purified by affinity and gel filtration chromatography. After concentration, the protein crystallized in space group P3121 with an unit cell with dimensions a=77 Å b=77 Å c=160 Å α=90° β=90° γ=120°. Csm2 was solved via cadmium single wavelength anomalous diffraction phasing at 2,4 Å resolution at a wavelength of 1,458 Å. The structure reveals that Csm2 is composed of a large 42 amino-acid long α-helix flanked by three shorter α-helices. The structure also shows that the protein is capable of forming dimers mainly via an extensive contact surface conferred by its long α-helix. This interaction is further stabilized by the N-terminal helix, which is inserted into the C-terminal helical portion of the adjacent subunit. The dimerization of Csm2 was additionally confirmed by size exclusion chromatography of the pure recombinant protein followed by mass spectrometry analysis of the eluted fractions. Because of its role in the Csm CRISPR RNP complex, the crystal structure of Csm2 is of great importance for clarifying the mechanism of action of the subtype IIIA CRISPR-Cas system, in the context of the different CRISPR-Cas systems.