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Incorporation de la beta alanine dans des polypeptides

Abstract : Living cells use 20 canonical α amino acids during ribosomal protein synthesis, although in rare cases, other amino acids such as selenocysteine or pyrrolysine can be used. The repertoire of usable amino acids is therefore quite limited. This limits the ability to build proteins having new properties. A dominant trend in the field of protein engineering is the construction of systems capable of incorporating non-canonical amino acids during in vivo protein synthesis. These studies have been very successful, but all amino acids incorporated until the 2010s were α-amino acids. Incorporation of β-amino acids at discrete sites would create unprecedented flexibility in the main chain, which would increase the potential for new protein folds. It has recently been shown using an in vitro protein synthesis system that it is possible to incorporate β-amino acids at specific positions (Katoh and Suga, 2018). In order to transpose this system in vivo, the main limitation is the aminoacylation of tRNAs with β -amino acids. It has recently been proposed that some aminoacyl-tRNA synthetases can use β-amino acids, but this capacity remains limited. The aim of this thesis is to incorporate β-methionine in polypeptides in vivo. For this purpose, we have characterized the recognition and use of L-β-homomethionine by methionyl-tRNA synthetase (MetRS) from E. coli. In particular, we have determined a high-resolution crystallographic structure of the MetRS:β-Met complex. Using fluorescence spectroscopy and mass spectroscopy, we were able to demonstrate the activation of -Met into adenylate as well as its esterification onto tRNAMet. However, the measured efficiencies are very small compared to what was published. Contamination of commercial β-Met with methionine may explain these differences. An in silico study based on the structure of the MetRS:β-Met complex was conducted in collaboration with the laboratory's bioinformatics team in order to search for mutant enzymes more efficient in the use of β-amino acids. Finally, we initiated the implementation of a directed evolution method to improve the efficiency of incorporation of amino acids in vivo.
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Submitted on : Wednesday, February 19, 2020 - 4:34:46 PM
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Giuliano Nigro. Incorporation de la beta alanine dans des polypeptides. Biochimie [q-bio.BM]. Université Paris Saclay (COmUE), 2019. Français. ⟨NNT : 2019SACLX064⟩. ⟨tel-02484790⟩



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