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Production of recombinant fragments of the transmembrane portions of the Na+/Ca2+ exchanger of Drosophila melanogaster for structural studies

Grant number: 14/20808-5
Support type:Scholarships in Brazil - Master
Effective date (Start): January 01, 2015
Effective date (End): August 31, 2016
Field of knowledge:Biological Sciences - Biochemistry - Chemistry of Macromolecules
Cooperation agreement: Coordination of Improvement of Higher Education Personnel (CAPES)
Principal researcher:Roberto Kopke Salinas
Grantee:Phelipe Augusto Mariano Vitale
Home Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil


Na+/Ca2+ exchangers (NCX) catalyze the counter transport of Na+ and Ca2+ across the plasma membrane. They are essential for the maintenance of the intracellular calcium homeostasis in different cell types. The NCX proteins consist of two transmembrane domains separated by a very large intracellular loop of approximately 500 amino acids. While the transmembrane region catalyzes the translocation of Na+ and Ca2+ across the lipid bilayer, the intracellular loop is responsible for the exchanger regulation. The binding of intracellular Ca2+ to the first intracellular Calcium-Binding Domain (CBD1) activates the NCX. However, in contrast to all other NCX, the Na+/Ca2+ exchanger of Drosophila melanogaster (CALX) is inhibited by the binding of calcium to CBD1. In spite of the physiological importance of the NCX, very little is known in structural details about how it functions and the mechanism of allosteric regulation. Thus, the goals of this project are to obtain further structural data about the NCX using the Drosophila's exchanger, CALX, as a model system. We propose to express and purify constructs corresponding to the transmembrane domains of the CALX or to the full-length protein. It is well recognized that it is very difficult to study the structure of membrane proteins by NMR or by X-ray crystallography. The CALX contains 10 transmembrane helices while the largest membrane protein ever studied by NMR has 7 transmembrane helices. For this reason, we propose to focus on the development of protocols in order to prepare good samples that would be suitable for future structural studies. (AU)

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