Functional and structural analysis of the glutamine ABC transporter from Mycobacterium tuberculosis

• Grant number: 14/19570-4
• Support Opportunities: Scholarships abroad - Research Internship - Post-doctor
• Effective date (Start): January 12, 2015
• Effective date (End): April 11, 2015
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andrea Balan Fernandes
• Grantee: Ana Leticia Gori Lusa
• Supervisor: Isabel Moraes
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Research place: Diamond Light Source, England
• Associated to the scholarship: 12/23469-1 - Production of ABC Transporters from Mycobacterium tuberculosis for Functional and Structural Studies - A Platform for Membrane Protein Production, BP.PD

Abstract

Membrane proteins corresponding to 25% to 30% of all transcribed sequences in known genomes and perform a range of essential functions in cellular metabolism. Substantial progress has been made in tackling many of the bottlenecks for expression, purification and determining of the crystal structures of alfa-helical membrane proteins. In addition, they represent 50% of the targets of the pharmaceutical industry. In M. tuberculosis, the causative agent of the tuberculosis, ATP-Binding Cassete (ABC) transporter family of membrane proteins is involved in drug transport and resistance allowing the development of the multi drug-resistant (MDR) and extensively drug-resistant (XDR) strains. Therefore, understanding efflux mechanisms is becoming increasingly important in the area of tuberculosis drug discovery. In the main project we aim to produce several ABC transporters from M. tuberculosis for functional and structural studies. After a set of experiments involving cloning, expression and purification trials of proteins forming the complexes, we selected three transporters for detailed characterization. Genes Rv2563 and Rv2564 encode a permease and an ATPase of the putative glutamine transporter, amino acid that is essential in the nitrogen pathway. The Rv2563 was previously expressed and crystallized in absence of the ATPase and crystals diffracted at 7 Å resolution. However, in order to get the full transporter efforts were done to express and purify the ATPase and the complex together. At the moment the complex was produced and preliminarly analysis revealed it is stable with DDM. In this sense, the objectives of this proposal are express and purify the complex in large scale for crystallization trials and diffraction experiments using the microfocus beamline of the Diamond Light Source. In addition we will express and try to obtain structural data from the periplasmic domain of the permease, which will be important for future phasing and determining of the crystal structure of the full complex. These experiments will be performed at the Membrane Protein Laboratory at Diamond Light Source under supervision of our collaborator Dr. Isabel de Moraes. (AU)