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Characterization of the molecular interactions within selenocysteine synthesis pathway proteins

Grant number: 14/16005-4
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): September 01, 2014
Effective date (End): July 31, 2015
Field of knowledge:Biological Sciences - Biophysics
Principal Investigator:Otavio Henrique Thiemann
Grantee:Jessica Fernandes Scortecci
Home Institution: Instituto de Física de São Carlos (IFSC). Universidade de São Paulo (USP). São Carlos , SP, Brazil
Associated research grant:08/57910-0 - National Institute of Structural Biotechnology and Medicinal Chemistry in Infectious Diseases, AP.TEM

Abstract

The existence of a greater variety of amino acids encoded by the genetic code has stimulated studies on the mechanisms of synthesis, recognition and incorporation of these residues in nascent polypeptide chains. As an example, we can highlight the specific incorporation of the amino acid selenocysteine, a co-translational event guided by the UGA codon. In bacteria, this pathway is formed by a complex molecular machinery composed of the proteins Selenocysteine Synthase (SELA), Recognition-Specific Elongation Factor (SELB), Selenophosphate Synthetase (SELD or SPS), a specific tRNA (SELC or tRNAsec), mRNA specific sequence (Sequence Insertion Selenocisteínas - SECIS), Seril tRNA Synthetase (SerRS), and other enzymes that supply the pathway with key substrates for the biosynthesis of selenocysteines such as Selenocysteine Lyase (CSDB). Because selenium has a high toxicity in the cellular environment, it is essential to understand the catalytic mechanism and elements involved in the formation of complexes. In bacteria, the interaction between CSDB and SPS was proposed in 2008, however, never verified experimentally. This project aims to the cloning, expression and purification of the enzyme CSDB involved in this specific interaction in addition to the characterization of CSDB interaction with SPS through techniques such as fluorescence anisotropy spectroscopy (FAS), differential scanning calorimetry (DSC) isothermal titration calorimetry (ITC) of the proteins proposed by the work of 2008. Attempts to the structural characterization using techniques such as X-rays scattering at low angles (SAXS) and crystallization trials will be conducted. With these results, we will contribue to the characterization of the incorporation of selenium in addition to providing models of interaction between proteins that are involved in metabolism of toxic elements.

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