Nature Chemical Biology
Nature Chemical Biology
5 years ago

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria

Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria
Anzhi Yao, Yuhan Zhang, Sean A Reed, Mingchao Kang, Peng-Yu Yang, Hui Guo, Tao Liu, Claudio Zambaldo, Peter G Schultz, Ian A Wilson, Renhe Liu, Xiaozhou Luo, Jintang Du, Weimin Xuan, Xiaoxuan Lyu, Xueyong Zhu, Rongsheng E Wang, Guangsen Fu, Feng Wang, Chunhui Huang
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.

Publisher URL: http://dx.doi.org/10.1038/nchembio.2405

DOI: 10.1038/nchembio.2405

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.