Structural and functional characterization of molecular chaperones Structural and functional characterization of molecular chaperones

Author(s)

Glaucia Melina Squizato Pinheiro

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Química
• Defense date: 2015-10-12

Examining board members

Carlos Henrique Inacio Ramos; Shaker Chuck Farah; Leandro Ramos Souza Barbosa; Fábio Cesar Gozzo; Taicia Pacheco Fill

• Advisor: Carlos Henrique Inacio Ramos

Abstract

Molecular chaperones belong to a family of proteins that are essential to maintain homeostasis since they are able to assist the formation of native structure of polypeptides and also to prevent incorrect folding and aggregation. The chaperones of the family `small heat shock proteins¿ (sHSPs) are expressed in several organisms such as bacteria, fungi, plants and animals, significantly under conditions of thermal stress, contributing to thermotolerance while protecting from aggregation. In plants, sHSPs are present in all cellular compartments, highlighting its importance. Due to the biotechnological relevance of sugarcane, we took to characterize structurally and functionally two os its sHSPs: the SsHSP21.5-mt from mitochondria and the SsHSP22.3-cp from chloroplast. The genes of these proteins were expressed in Escherichia coli and the recombinant proteins were isolated and purified, by two chromatographic steps, with high purity (> 95%) and in a single oligomeric state. Circular dichroism and fluorescence spectroscopy analyses showed that both proteins were folded, presenting predominantly ?-sheet secondary structure. The hydrodynamic characterization determined the molecular weight, the diffusion coefficient and the Stokes radius of the proteins in solution, revealing that both chaperones form similar large oligomers in solution with spherical shapes. However, the chaperone activity was not equivalent, since SsHSP22.3-cp was most effective in protecting all substrate models (malate dehydrogenase, citrate synthase, insulin, lysozyme and extract of E. coli) when compared to SsHSP21. 5-mt. These results showed that the two sHSPs were structurally similar, but had distinct chaperone activity, probably due to the low identity among the N-terminal domains, which are known to be involved with substrate affinity. These results confirm the importance of having great diversity of sHSP chaperones in plants for homeostasis maintenance (AU)

• FAPESP's process: 11/05115-5 - Cloning, expression and study of the relationship between structure and function of molecular chaperones thermosensitive sugarcane
• Grantee: Glaucia Melina Squizato Pinheiro de Castro
• Support Opportunities: Scholarships in Brazil - Doctorate