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Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria
Luo, X Fu, G Wang, RE Zhu, X Zambaldo, C Liu, R Liu, T Lyu, X Du, J Xuan, W
Luo, X
Fu, G
Wang, RE
Zhu, X
Zambaldo, C
Liu, R
Liu, T
Lyu, X
Du, J
Xuan, W
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Post-print
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2017-08-01
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10.1038/nchembio.2405
Abstract
© 2017 Nature America, Inc., part of Springer Nature. All rights reserved. Tyrosine phosphorylation is a common protein post-translational modification that plays a critical role in signal transduction and the regulation of many cellular processes. Using a propeptide strategy to increase cellular uptake of O-phosphotyrosine (pTyr) and its nonhydrolyzable analog 4-phosphomethyl-L-phenylalanine (Pmp), we identified an orthogonal aminoacyl-tRNA synthetase-tRNA pair that allows site-specific incorporation of both pTyr and Pmp into recombinant proteins in response to the amber stop codon in Escherichia coli in good yields. The X-ray structure of the synthetase reveals a reconfigured substrate-binding site, formed by nonconservative mutations and substantial local structural perturbations. We demonstrate the utility of this method by introducing Pmp into a putative phosphorylation site and determining the affinities of the individual variants for the substrate 3BP2. In summary, this work provides a useful recombinant tool to dissect the biological functions of tyrosine phosphorylation at specific sites in the proteome.
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Springer Science and Business Media LLC
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Nature Chemical Biology
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