Download MendeleyGenetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.
Luo, X., Fu, G., Wang, R.E., Zhu, X., Zambaldo, C., Liu, R., Liu, T., Lyu, X., Du, J., Xuan, W., Yao, A., Reed, S.A., Kang, M., Zhang, Y., Guo, H., Huang, C., Yang, P.Y., Wilson, I.A., Schultz, P.G., Wang, F.(2017) Nat Chem Biol 13: 845-849
- PubMed: 28604693
- DOI: https://doi.org/10.1038/nchembio.2405
- Primary Citation of Related Structures:
5U36 - PubMed Abstract:
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.
Organizational Affiliation:
Department of Chemistry, The Scripps Research Institute, La Jolla, California, USA.
