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Modélisation de l'évolution dirigée de la tyrosyl-ARNt synthétase in silico. Introduction d'acides aminés non naturels dans les protéines.

Abstract : Although most proteins are usually synthesized with L-amino acids, D-amino acids are present in organisms in their free states, included in shorts peptides and even in particular proteins. Introducing D-amino acids in proteins in a controlled way can provide a biotechnological advantage, adding new function to proteins. Tyrosyl-tRNA synthetase (TyrRS) has already been used to incorporate more than thirty tyrosine analogues in proteins. Using the D-enantiomer of the tyrosine can give resistance to proteases in cells. Because proteases recognizes less proteins with such amino-acids, peptides bearing those will stay active much longer. TyrRS has a natural though weak activity for D-Tyrosine. The purpose of this thesis is to improve this activity by an in silico directed evolution method. The method consists in randomly mutating a protein to select mutants with the desired properties, mimicking mechanism of natural evolution. We use for this study an in house program of directed evolution. It is based on an iterative optimization procedure of both protein sequence and structure. Some mutants have been predicted by this method and proposed to biochemists for experimental in vivo validations. They find that some mutants present a weak, but measurable, activity for the D-Tyrosine. The field of study has thus been enlarged to other mutation positions around the tyrosine ammonium group. We study TyrRS and its binding with either L or D tyrosine and furthermore consider an activated form of the aminoacid, the tyrosinyl-adenylate in both geometry L and D. This compound has been modeled with the E. coli TyrRS to make molecular dynamic studies. We have use the large ressources of the CINES (Centre Informatique National de l'Education Supérieure) and our laboratory cluster to simulate a maximum of molecular dynamics on our predicted mutants. Another goal of this work was to improve our in house protein design methods with a more sophisticated solvent model. In order to do so, we used Born's model based on the Poisson-Boltzmann theory. Rather than describing solvent as individual molecules, this model considers it as an electrostatic continuum surrounding the protein. We validated that new protocol on samples presenting experimental data to compare with. We succesfully tested three small peptides before further simulationss were carried out in the same way on a Bacillus stearothermophilus TyrRS mutants set.
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https://pastel.archives-ouvertes.fr/pastel-00622571
Contributor : Najette Amara <>
Submitted on : Monday, September 12, 2011 - 3:18:08 PM
Last modification on : Wednesday, July 29, 2020 - 4:10:05 PM
Long-term archiving on: : Sunday, December 4, 2016 - 1:20:20 PM

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  • HAL Id : pastel-00622571, version 1

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Najette Amara. Modélisation de l'évolution dirigée de la tyrosyl-ARNt synthétase in silico. Introduction d'acides aminés non naturels dans les protéines.. Bio-Informatique, Biologie Systémique [q-bio.QM]. Ecole Polytechnique X, 2011. Français. ⟨pastel-00622571⟩

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