Project Summary Healthy and diseased physiological states are governed by a complex web of interacting proteins that confer the collective behavior observed in cells. These protein networks are decorated with posttranslational modifications (PTMs) that determine their structure, function, and impart specificity for cellular signaling. Phosphorylation and acetylation represent two common PTMs that dictate healthy and disease states in human cells. For instance, 14-3-3 proteins scaffold thousands of important phosphoproteins with evidence suggesting that acetylation can modify its function. Current progress toward the elucidation of PTM-mediated signaling networks is hampered by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific combinations of modified amino acids. Our previous efforts utilized a recoded E. coli strain (i.e., genomically recoded organism) to synthesize all human phosphoserine proteins using a genetic code expansion technique. We expanded this work through the development of a two hybrid like technology, named HI-P. HI-P validated previously observed phosphorylation dependent protein-protein interactions and identified scores of novel phosphoserine-mediated interactions across the human proteome that have been validated in biochemical- and cell-based assays. Our approach allows for synthetic DNA inputs to direct ribosome-based phosphoprotein synthesis and thus creates a programmable genetic tool to study the human phosphoproteome at the molecular level. Since deciphering complex protein networks require studying the impact of multiple PTMs in isolation and in combination, the key contribution of the proposed research is expected to expand the ability to genetically encode phosphoserine and acetylation at precise positions in 14-3-3 proteins to reveal PTM-mediated protein- protein interactions. Specific Aims: In this proposal, we seek to leverage a strong foundation of technologies, expertise, and preliminary data to construct a recoded E. coli with a single stop codon (two open codons) (Aim 1), develop a protein synthesis system capable of simultaneous encoding of phosphorylated and acetylated amino acids into proteins (Aim 2), and employ these capabilities to deconvolute PTM-mediated 14-3-3 protein network interactions (Aim 3). Significance: This work will be significant because it will enable the expression of programmable human proteins containing two PTMs thereby establishing a new approach to decipher complex human signaling networks at the molecular level. We anticipate this work will elucidate novel 14-3-3 protein network interactions governed by PTMs and enable new research into biomolecular and protein mechanisms that can be used to develop new therapies for human disease.