Understanding the influence of artificial amino acid side chains containing fluorine atoms on peptide or protein properties is undisputed important for peptide and protein engineering. For this purpose, incorporation of fluorinated amino acids (fAAs) has been applied in vitro by Solid-Phase Peptide Synthesis (SPPS), and in vivo by bacterial ribosomal translation machinery.
This study focused on the latter method, which allows the synthesis of fluorinated proteins with high fidelity, and no limitation in length. The wild type isoleucyl-tRNA synthetase (IleRS) from Escherichia coli has been used to investigate the interaction of aminoacylation and editing activities against fAAs, including small aliphatic AAs as (2S)-4,4,4-trifluoroethylglycine (TfeGly). Although IleRS activates TfeGly, we initially could not achieved the bacterial production of fluorinated peptides and proteins. The reason behind this failed attempt is the efficient editing against TfeGly, which leads to reduced dissociation rate of TfeGly-tRNAIle from the CP1 domain of IleRS and prevents the accummulation of TfeGly-tRNAIle. Taking this knowledge in to account, we used a post-transfer editing deficient mutant of IleRS, IleRSAla10, and succeeded in ribosomal incorporation of TfeGly.
By this detailed analysis of each step of ribosome-mediated translation process, we are able to provide valuable data for protein engineering towards fluorination of peptides and proteins.
J.-S. Völler, M. Dulic, U. I. M. Gerling-Driessen, H. Biava, T. Baumann, N. Budisa, I. Gruic-Sovulj, B. Koksch, Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation, ACS Cent. Sci., 2017, 3 (1), 73–80.