Serine phosphorylation is a key post-translational modification that regulates diverse biological processes. Powerful analytical methods have identified thousands of phosphorylation sites, but many of their functions remain to be deciphered. A key to understanding the function of protein phosphorylation is access to phosphorylated proteins, but this is often challenging or impossible. Here we evolve an orthogonal aminoacyl-tRNA synthetase/tRNA CUA pair that directs the efficient incorporation of phosphoserine into recombinant proteins in E. coli. Moreover, combining the orthogonal pair with a metabolically engineered E. coli enables the site-specific incorporation of a non-hydrolyzable analog of phosphoserine. Our approach enables quantitative decoding of the amber stop codon as phosphoserine and we purify several milligrams-per-liter of proteins bearing biologically relevant phosphorylations that were previously challenging or impossible to access: including phosphorylated ubiquitin and a kinase (Nek7) that is synthetically activated by a genetically encoded phosphorylation in its activation loop.The phosphorylation of proteins in eukaryotic cells is a key post-translational modification that regulates essential and diverse biological phenomena. Phosphorylation is installed by protein kinases on serine, threonine, tyrosine and histidine residues, and removed by phosphatases, and serine phosphorylation is the most abundant phosphorylation observed in Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer.

Deoxyribozymes (DNA catalysts) have been reported for cleavage of RNA phosphodiester linkages, but cleaving peptide or DNA phosphodiester linkages is much more challenging. Using in vitro selection, here we identified deoxyribozymes that sequence-specifically hydrolyze DNA with multiple turnover and rate enhancement of 108 (possibly as high as 1014). The new DNA catalysts require both Mn2+ and Zn2+, which is intriguing because many natural DNA nucleases are bimetallic protein enzymes.