Application of synthetic copolymer scaffolds for cryo-electron microscopy studies of the human mitochondrial pyruvate carrier

• Grant number: 19/02261-2
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate (Direct)
• Start date: May 01, 2019
• End date: October 10, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Chemistry of Macromolecules
• Principal Investigator: Andre Luis Berteli Ambrosio
• Grantee: José Edwin Neciosup Quesñay
• Supervisor: Timothy Dafforn
• Host Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
• Institution abroad: University of Birmingham, England
• Associated to the scholarship: 15/02734-7 - Functional and structural studies of four homologous recombinants of the mitochondrial pyruvate carrier complex, BP.DD

Abstract

The determination of the molecular structure of membrane proteins by cryo-electron microscopy (cryo-EM) represents the current frontier in the field of structural biology. Recent advances in detector technologies and the development of powerful maximum likelihood image processing routines have allowed for the routine description of high-resolution structures of biomolecules (> 4 Å); this collection of accomplishments signaled the beginning of a new era in molecular biology, where structures at near-atomic resolution are no longer the privilege of X-ray crystallography. Primary challenges are most currently related to sample preparation; more specifically, the detergent-free extraction and purification of the protein samples of interest and the identification of appropriate freezing conditions during grid preparation. In the past few years - and funded mainly by FAPESP - our laboratory gained substantial experience in the purification and functional reconstitution of human mitochondrial membrane proteins, which is ready for structural studies. To this end, we now seek to gain hands-on knowledge in approaches related to cryo-EM and propose a collaboration with the laboratory of Dr. Timothy Dafforn (University of Birmingham, UK) with the goal of overcoming the challenges above, by making use of synthetic copolymers. Dr. Dafforn is the pioneer in the revolutionary use of styrene-maleic acid copolymers that allows for the direct and straightforward extraction of membrane proteins from its native membrane system. Once we overcome these challenges, the micrograph dataset used for structure determination will be collected at the Brazilian National Laboratory of Nanotechnology (LNNano), in Campinas, SP, which will soon host a state-of-the-art Titan Krios electron microscope, equipped with a phase plate and a direct electron detector, among other accessories. (AU)

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

QUESNAY, JOSE EDWIN NECIOSUP; POLLOCK, NAOMI L.; NAGAMPALLI, RAGHAVENDRA SASHI KRISHNA; LEE, SARAH C.; BALAKRISHNAN, VIJAYAKUMAR; DIAS, SANDRA MARTHA GOMES; MORAES, ISABEL; DAFFORN, TIM R.; AMBROSIO, ANDRE LUIS BERTELI. Insights on the Quest for the Structure-Function Relationship of the Mitochondrial Pyruvate Carrier. BIOLOGY-BASEL, v. 9, n. 11, NOV 2020. (14/20673-2, 17/11766-5, 19/02261-2, 13/07600-3, 14/06954-9, 18/00492-4, 17/02391-8, 15/02734-7)