An evolved aminoacyl-tRNA synthetase with atypical polysubstrate specificity |
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Authors: | Young Douglas D Young Travis S Jahnz Michael Ahmad Insha Spraggon Glen Schultz Peter G |
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Institution: | Department of Chemistry and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, United States. |
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Abstract: | 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. |
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