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Functional and structural characterization of the glutamine transporter from Mycobacterium tuberculosis

Grant number: 15/17476-3
Support type:Scholarships in Brazil - Doctorate (Direct)
Effective date (Start): March 01, 2016
Effective date (End): May 31, 2021
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Andrea Balan Fernandes
Grantee:Marcelo de Cássio Barreto de Oliveira
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
Associated scholarship(s):18/26740-4 - Functional and structural analysis of the ABC transporter Rv2563/Rv2564 from Mycobacterium tuberculosis, BE.EP.DD

Abstract

Membrane proteins are considered the new challenges of Structural Biology given their medical relevance. They represent more than 50% of the targets of the pharmaceutical industry and about 30% of genomes from different organisms. With the aim to set up a platform for the expression and production of membrane proteins in our laboratory for use in functional and structural studies, we have chosen as targets a set of ATP-Binding Cassete transporters from Mycobacterium tuberculosis. ABC transporters have great medical and pharmaceutical relevance as they are related to the development of Multiple Drug Resistance phenomenon (MDR) in cancer treatment and bacterial resistance, including in tuberculosis. Mycobacterium tuberculosis is the causative agent of tuberculosis and about 3% of its genome encode ABC transporters. Although identified, there are no functional studies of the large majority of these transporters opening a possibility of characterizing their functions, and defining the range of substrates as a step for developing new forms of disease control. In this sense, our group has studied the glutamine transporter from M. tuberclosis, which is encoded by genes Rv2563 (permease) and Rv2564 (ATPase). During the post-doctoral project developed by Dr. Ana Leticia Lusa (Proc. FAPESP 2012/23469-1), the complex permease/ATPase was expressed and purified in the presence of detergents and submitted to spectroscopic assays and crystallization, which generated crystals that diffracted with a maximum resolution of 5 Å. In the analyzes of bioinformatics, Rv2563 permease presented an additional 196 residues periplasmic domain, which we believe to perform an essential role in the capture of glutamine. In order to keep the functional and structural characterization of this transporter, this project aims to characterize the periplasmic domain (Rv2563_per) and ATPase, as well as the production of permease (Rv2563) and ATPase (Rv2564) in complex. Protocols of expression and purification of complex are already established and they must be optimized to increase the quality of crystals. Following expression and purification of the domains separately, they will be subjected to spectroscopic assays to measure the interaction with glutamine and other amino acids, ATP and ATPase inhibitors, and co-crystallization trials in the presence of ligands. The results of this project will be of scientific importance since there is no structural or... (AU)