Genetically encoding phosphotyrosine and its nonhydrolyzable analog in bacteria.
Nat Chem Biol
; 13(8): 845-849, 2017 Aug.
Article
in En
| MEDLINE
| ID: mdl-28604693
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.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Codon, Nonsense
/
Phosphotyrosine
/
Escherichia coli
Language:
En
Journal:
Nat Chem Biol
Journal subject:
BIOLOGIA
/
QUIMICA
Year:
2017
Document type:
Article
Affiliation country: