Journal
NATURE CHEMICAL BIOLOGY
Volume 13, Issue 8, Pages 845-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nchembio.2405
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Funding
- DOE, Basic Energy Sciences, Office of Science [DE-AC02-06CH11357]
- NCI [Y1-CO-1020]
- NIGMS [Y1-GM-1104]
- NIH [5R01 GM062159-14]
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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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