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Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine

Titelangaben

Baumann, Tobias ; Hauf, Matthias ; Richter, Florian ; Albers, Suki ; Möglich, Andreas ; Ignatova, Zoya ; Budisa, Nediljko:
Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine.
In: International Journal of Molecular Sciences. Bd. 20 (2019) Heft 9 . - 2343.
ISSN 1422-0067
DOI: https://doi.org/10.3390/ijms20092343

Angaben zu Projekten

Projektfinanzierung: Alexander von Humboldt-Stiftung
Deutsche Forschungsgemeinschaft

Abstract

Engineering aminoacyl-tRNA synthetases (aaRSs) provides access to the ribosomal incorporation of noncanonical amino acids via genetic code expansion. Conventional targeted mutagenesis libraries with 5–7 positions randomized cover only marginal fractions of the vast sequence space formed by up to 30 active site residues. This frequently results in selection of weakly active enzymes. To overcome this limitation, we use computational enzyme design to generate a focused library of aaRS variants. For aaRS enzyme redesign, photocaged ortho-nitrobenzyl tyrosine (ONBY) was chosen as substrate due to commercial availability and its diverse applications. Diversifying 17 first- and second-shell sites and performing conventional aaRS positive and negative selection resulted in a high-activity aaRS. This MjTyrRS variant carries ten mutations and outperforms previously reported ONBY-specific aaRS variants isolated from traditional libraries. In response to a single in-frame amber stop codon, it mediates the in vivo incorporation of ONBY with an efficiency matching that of the wild type MjTyrRS enzyme acylating cognate tyrosine. These results exemplify an improved general strategy for aaRS library design and engineering.

Weitere Angaben

Publikationsform: Artikel in einer Zeitschrift
Begutachteter Beitrag: Ja
Keywords: directed evolution; enzyme design; gene libraries; genetic code expansion; mutagenesis; noncanonical amino acids; protein engineering; protein modification; unnatural amino acids
Institutionen der Universität: Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie > Lehrstuhl Biochemie II - Photobiochemie - Univ.-Prof. Dr. Andreas Möglich
Fakultäten
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie
Titel an der UBT entstanden: Nein
Themengebiete aus DDC: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
Eingestellt am: 12 Jul 2019 09:02
Letzte Änderung: 12 Dec 2023 13:03
URI: https://eref.uni-bayreuth.de/id/eprint/51418