Analysis and characterization of the n- and C- terminal domains of human BiP protein (HspA5)

• Grant number: 19/14209-5
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date until: November 01, 2019
• End date until: October 31, 2020
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Julio Cesar Borges
• Grantee: Bruna Siebeneichler
• Host Institution: Instituto de Química de São Carlos (IQSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Associated research grant: 17/26131-5 - The chaperome: study of the relationship of the structure of its components and the maintenance of proteostasis, AP.TEM

Abstract

The binding immunoglobulin protein (BiP) belongs to the class of proteins called molecular chaperones and to the family of 70 kDa heat shock proteins (Hsp70). This macromolecule is mainly found in the endoplasmic reticulum (RE) and acts in the control of cellular proteostasis, interacting with proteins, called client proteins, so that they acquire their native conformation. It has two domains, the N-terminal or nucleotide-binding domain (NBD) and the C-terminal domain or client protein binding (PBD). BiP plays an important role in regulating ER response to different types of cellular stress that may be associated with neurodegenerative diseases and cancer. Given its importance, this research proposal aims to study the BiP's NBD and PBD recombinant proteins taking into account their structural and functional properties. In this context, the recombinant domains will be purified by chromatographic techniques and characterized by biophysical techniques such as circular dichroism spectropolarimetry (CD), intrinsic tryptophan fluorescence and analytical size exclusion chromatography. In addition, the domains will have their chemical and thermal stability monitored by differential scanning calorimetry (DSC), CD and fluorescence. Further, the N and C-terminal domains will be functionally analyzed by their ATPase activity and protein client aggregation prevention assays, respectively. The obtained data will allow to characterize in terms of structure and function the PBD and NBD of the BiP, also in comparison to the recombinant human BiP, as well as to understand the importance and contribution of the same to the functional performance of BiP. (AU)