Design, Synthesis and Catalytic Activity of Protein Containing Thiotyrosine as an Active Site Residue

Author:

Bachelart Thomas1,Kumar Shailesh1,Jouin Alexis1,Yousef Mo'ath1,Kieffer Bruno2,Torbeev Vladimir1ORCID

Affiliation:

1. École Supérieure de Biotechnologie de Strasbourg (ESBS) CNRS UMR 7242 Biotechnology and Cellular Signalling University of Strasbourg 67400 Illkirch France

2. Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC) CNRS UMR 7104, INSERM U 1258 University of Strasbourg 67400 Illkirch France

Abstract

AbstractNative chemical ligation is a key reaction in the toolbox of chemical methods for the synthesis of native and modified proteins. The catalysis of ligation is commonly performed by using small aryl‐thiol molecules added at high concentrations. In this work, we incorporated thiotyrosine, a non‐canonical amino acid containing an aryl‐thiol moiety, into a designed cyclic protein « sans queue ni tête ». Importantly, the protein environment reduced the pKa of the thiol group to 5.8–5.9, which is significantly lower than the previously reported value for thiotyrosine in a short peptide (pKa 6.4). Furthermore, we demonstrated the catalytic activity of this protein both as hydrolase and in native chemical ligation of peptides. These results will be useful for the development of efficient protein catalysts (enzymes) for protein synthesis and modification.

Funder

HORIZON EUROPE European Research Council

Publisher

Wiley

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