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Evolution dirigée de deux aminoacyl-ARNt synthétases : Mise en place et applications d'une méthode de 'protein design'.

Abstract : Protein design aims to develop new proteins with new structural and/or functional properties. The principle is to identify among the available sequences, those that preserve the protein's three dimensional fold and that confer the desired properties. The general procedure can be decomposed into two steps : (i) the interaction energy between all the residue pairs is precomputed and stored in an energy matrix taking into account all amino acid types and all possible conformations, (ii) an optimization algorithm explores simultaneously sequence and conformational space to determine the best amino acid combination. Next, different filters (based on affinity, protein stability...) can be applied to separate functional sequences (for a given fold) from the non functional ones. We first focused on the development of the protein design procedure, particularly, on the setting up and optimization of the energy function and the implementation of the optimization algorithm. We have shown that our procedure is robust because it performs well for a wide variety of applications crucial in protein design such as: prediction of sidechain orientation, prediction of stability or affinity changes due to point mutations and design of native-like sequences for a set of globular proteins. For all of these applications the quality of results is competitive with those obtained by other groups. Next, we applied our procedure to more complex systems such as protein:ligand complexes. We focused on aspartyl-tRNA synthetase (AspRS) and asparaginyl-tRNA synthetase (AsnRS). These enzymes play a crucial role in preserving the accuracy of genetic code translation, linking their specific amino acid to a cognate tRNA, which carries the corresponding anticodon. First, we performed the design of the whole active site of AspRS and AsnRS in presence of their specific or non specific ligands in order to test the performance of our procedure. The quality of the designed sequences is consistent with those observed on entire globular proteins. On the other hand, we showed that our procedure was sensitive to the nature of the ligand present in the active site. Finally, we performed the design of a limited number of selected positions of the AsnRS active site so that the synthetase can bind preferentially aspartate over asparagine. A set of promising mutants has been retained. Their stability and affinity for native and non-native ligands are now analyzed by molecular dynamics.
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Submitted on : Friday, July 23, 2010 - 9:16:32 AM
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  • HAL Id : pastel-00003713, version 1



Anne Lopes. Evolution dirigée de deux aminoacyl-ARNt synthétases : Mise en place et applications d'une méthode de 'protein design'.. Biologie cellulaire. Ecole Polytechnique X, 2008. Français. ⟨pastel-00003713⟩



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