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Structural studies of fragments derived from the Na+/Ca2+ exchanger by solution NMR

Grant number: 15/26356-1
Support Opportunities:Scholarships in Brazil - Master
Start date: April 01, 2016
End date: July 31, 2017
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Principal Investigator:Roberto Kopke Salinas
Grantee:Tatiana Comporte Stabelini
Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Membrane proteins are involved in essential physiological processes such as the maintenance of the ionic balance, and intracellular signaling. Despite their involvement in numerous important physiological processes and of great pharmaceutical interest, structural studies of membrane proteins are still an expensive procedure, and are much more complex than structural studies of globular proteins. Na+/Ca2+ exchangers (NCX) form a family of membrane transporters that operate in the maintenance of intracellular Ca2+ homeostasis, and that are involved in pathological processes such as heart disease. The NCXs are present in several species of mammals (NCXs) and insects such as Drosophila melanogaster (CALX). These exchangers have two domains of 5 transmembrane helices (TMH) each. The TMH domains are responsible for promoting the specific transport of Ca2+ and Na+ through the lipid bilayer. A large cytoplasmic loop between TMH5 and TMH6 is responsible for regulating the exchanger activity. This loop has two sensor domains for intracellular Ca2 + (CBD1 and CBD2). The CALX exchanger is inhibited by Ca2+ binding to CBD1, while the NCXs are activated. The structure of the regions that connect CBD1 and CBD2 to the transmembrane helices 5 and 6 is unknown. The amino acid sequences near the TMH5 and TMH6 are fairly conserved, rich in hydrophobic and positively charged (TMH5) or negatively charged (TMH6) residues, and are potentially involved in the ionic regulation mechanism. In this work we chose to study two regions of the CALX exchanger: the TMH5 and TMH6 and their respective juxta-membrane regions. In the absence of structural data about the full length protein, the study of these fragments may help to understand the structure and behaviour of the complete exchanger. Different ways of expressing these fragments in E. coli and in cell free, and different membrane mimetic systems such as detergents, lipids and amphipathic polymers, will be tested throughout this study.

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Academic Publications
(References retrieved automatically from State of São Paulo Research Institutions)
STABELINI, Tatiana Comporte. Structural studies of fragments derived from the Na+/Ca2+ exchanger by solution NMR. 2018. Master's Dissertation - Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) São Paulo.