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Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine

Title data

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. Vol. 20 (2019) Issue 9 . - p. 2343.
ISSN 1422-0067
DOI: https://doi.org/10.3390/ijms20092343

Project information

Project financing: Alexander von Humboldt-Stiftung
Deutsche Forschungsgemeinschaft

Abstract in another language

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.

Further data

Item Type: Article in a journal
Refereed: Yes
Keywords: directed evolution; enzyme design; gene libraries; genetic code expansion; mutagenesis; noncanonical amino acids; protein engineering; protein modification; unnatural amino acids
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Andreas Möglich
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: No
DDC Subjects: 500 Science > 570 Life sciences, biology
Date Deposited: 12 Jul 2019 09:02
Last Modified: 12 Jul 2019 09:02
URI: https://eref.uni-bayreuth.de/id/eprint/51418