Project Summary This proposed work seeks to develop a suite of enabling technologies capable of producing synthetic phosphoproteins with the goal of transforming the field of human protein signaling from one that is purely observation‐based into one that biosynthesizes designer proteins to achieve a comprehensive understanding of complex signaling networks. The importance of phosphorylation is emphasized by the fact that phosphorylated proteins control most aspects of normal cellular homeostasis. Aberrations in protein phosphorylation can drive cancer, hypertension, diabetes, and neurodegenerative disorders. Thus, understanding differential patterns of protein phosphorylation in disease states is of extreme physiological and clinical interest. Analysis of phosphorylated amino acid residues has been limited by our inability to control these chemical modifications due to a lack of phosphomimetics that fully recapitulate the chemistry of phosphorylated residues. Current progress toward the elucidation of phospho‐signaling networks is hampered by the lack of methods to produce proteins containing specific combinations of phosphorylated amino acids. In particular, synthetic chemistry is inadequate for total phosphoprotein synthesis, and conventional biological methods do not control phosphorylation levels. We have recently developed a new technology, albeit limited to phosphoserine (pSer), that enables the synthesis of recombinant phosphoproteins. This technology directs phosphorylated amino acids into their physiologically relevant positions within proteins yet our functional understanding of protein phosphorylation will remain incomplete without access to phosphotyrosine (pTyr) and phosphothreonine (pThr) containing proteins. Specific Aims: In Aim 1, we will utilize mutagenesis and laboratory evolution to engineer an optimized tyrosyl aminoacyl‐tRNA synthetase for phosphotyrosine. In Aim 2, we will provide a solution to this problem by engineering an aminoacyl‐tRNA synthetase that can charge a phosphothreonine onto a special tRNA that reads a dedicated open codon. Unique to our approach, we will also employ our genomically recoded E. coli cells in which open stop codons can be converted into new sense codons that encode pThr and pTyr into precise locations in recombinant proteins. Significance: The overall outcome of our studies will be an enabling technology for the expression of pTyr and pThr containing proteins that will broadly enable research into disease mechanisms and can be used directly to develop new therapies for human disease. This will be the first technology able to re‐create human disease networks that are “difficult” or “impossible” to infiltrate, and will establish the paradigm for addressing other post‐translational modifications. More broadly, the proposed work will enable the re‐design of programmable signaling networks comprising proteins with natural and synthetic nonstandard amino acids capabl...