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An Evolved Aminoacyl-tRNA Synthetase with Atypical Polysubstrate Specificity,

2011; American Chemical Society; Volume: 50; Issue: 11 Linguagem: Inglês

10.1021/bi101929e

1943-295X

Douglas D. Young, Travis S. Young, Michael Jahnz, Insha Ahmad, Glen Spraggon, Peter G. Schultz,

Chemical Synthesis and Analysis

We have employed a rapid fluorescence-based screen to assess the polyspecificity of several aminoacyl-tRNA synthetases (aaRSs) against an array of unnatural amino acids. We discovered that a p-cyanophenylalanine specific aminoacyl-tRNA synthetase (pCNF-RS) has high substrate permissivity for unnatural amino acids, while maintaining its ability to discriminate against the 20 canonical amino acids. This orthogonal pCNF-RS, together with its cognate amber nonsense suppressor tRNA, is able to selectively incorporate 18 unnatural amino acids into proteins, including trifluoroketone-, alkynyl-, and halogen-substituted amino acids. In an attempt to improve our understanding of this polyspecificity, the X-ray crystal structure of the aaRS−p-cyanophenylalanine complex was determined. A comparison of this structure with those of other mutant aaRSs showed that both binding site size and other more subtle features control substrate polyspecificity.