Discovery and Investigation of Natural Editing Function against Artificial Amino Acids in Protein Translation.
Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) fromEscherichia colito investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNAIlein the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNAIlefrom IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.
Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universität Berlin, Takustrasse 3, 14195 Berlin, Germany; Department of Chemistry, Technische Universität Berlin, Müller-Breslau-Strasse 10, 10623 Berlin, Germany.
Jan-Stefan
JS
Völler
Department of Chemistry, Faculty of Science, University of Zagreb , Horvatovac 102a, 10000 Zagreb, Croatia.
Morana
M
Dulic
Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universität Berlin , Takustrasse 3, 14195 Berlin, Germany.
Ulla I M
UI
Gerling-Driessen
Department of Chemistry, Technische Universität Berlin , Müller-Breslau-Strasse 10, 10623 Berlin, Germany.
Hernan
H
Biava
Department of Chemistry, Technische Universität Berlin , Müller-Breslau-Strasse 10, 10623 Berlin, Germany.
Tobias
T
Baumann
Department of Chemistry, Technische Universität Berlin , Müller-Breslau-Strasse 10, 10623 Berlin, Germany.
Nediljko
N
Budisa
Department of Chemistry, Faculty of Science, University of Zagreb , Horvatovac 102a, 10000 Zagreb, Croatia.
Ita
I
Gruic-Sovulj
Institute of Chemistry and Biochemistry - Organic Chemistry, Freie Universität Berlin , Takustrasse 3, 14195 Berlin, Germany.
Beate
B
Koksch