Expressão, purificação e caracterização estrutural dos mutantes truncados da Hsp70-escort protein 1 humana

• Autor(a) principal: Gomes, Maria Júlia Mattos
• Data de Publicação: 2024
• Tipo de documento: Trabalho de conclusão de curso
• Idioma: por
• Título da fonte: Repositório Institucional da UFSCAR
• Texto Completo: https://repositorio.ufscar.br/handle/ufscar/19430
• topic: Chaperonas moleculares; Hsp70; Proteínas recombinantes; Caracterização biofísica; Molecular chaperones; Recombinant proteins; Biophysical characterization; CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS

The 70 kDa Heat shock protein (Hsp70 or HSPA in humans) is a highly conserved family of molecular chaperones that is expressed in various cellular compartments and is associated with several pathologies, including neurodegenerative diseases and cancer. Among HSPAs, the mitochondrial HSPA (HSPA9 or mortalin) plays a crucial role in the import of proteins synthesized in the cytosol into the mitochondria. This function requires interaction with a specific co-chaperone called human Hsp70-escort protein 1 (hHep1), whose L-shaped structure, containing a "zinc finger" domain, is fundamental for such interactions. hHep1 holds great importance in the maintenance of HSPA9 and HSPA1A (human Hsp701A) solubility, as well as the remodeling and stimulation of their ATPase activity. It is present in both the mitochondria and the cell nucleus, where it interacts with HSPAs such as HSPA1A. Due to its role in HSPA homeostasis, this study aimed to express, purify, and characterize hHep1 mutants with truncated N- and C-terminal regions to identify the primary regions responsible for its function. The hHep1-Core mutant, containing the "zinc finger" domain, was also studied to elucidate its structural and functional roles. The secondary and tertiary structures of the hHep1-Ndel, hHep1-Cdel, and hHep1-Core mutants were investigated using circular dichroism and fluorescence spectroscopy, respectively. The results revealed similarities in structure between the hHep1-Ndel and hHep1Δmts mutants, as well as between the hHep1-Cdel and hHep1-Core mutants. Moreover, the stability of the proteins was demonstrated for up to 72 hours, and their resistance to denaturing agents was demonstrated, with Cm values of 1.4 M, 1.9 M, and 1.5 M for hHep1-Ndel, hHep1-Cdel, and hHep1-Core, respectively. The size and shape of the mutants were also analyzed using analytical size exclusion chromatography, revealing that all proteins were monomeric, and that hHep1-Core had a more globular structure compared to the complete structure. The study concluded that the methods and conditions used were suitable for the expression, purification, and characterization of these recombinant proteins, providing a valuable contribution to the field of research.