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RSS FeedsIJMS, Vol. 20, Pages 2343: Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine (International Journal of Molecular Sciences)

 
 

11 may 2019 11:01:19

 
IJMS, Vol. 20, Pages 2343: Computational Aminoacyl-tRNA Synthetase Library Design for Photocaged Tyrosine (International Journal of Molecular Sciences)
 




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.


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138 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
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