Engineering a recoded organism to discover PTM-mediated protein binders

Yale University, New Haven CT

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Abstract

PROJECT SUMMARY Dissecting protein-protein interactions (PPI) at a systems level is essential in attaining a global view and a molecular understanding of the web of interdependent events that govern collective cellular behavior and human physiology in health and disease. The precise placement and chemical composition of post-translational modifications (PTMs) decorated across proteins determines their structure, function, and impart specificity for cellular signaling. Current progress toward the elucidation of PTM-mediated signaling and function is hampered by the challenge of studying transient PTMs in cells and limited methods to produce proteins containing specific combinations of modified amino acids. Recent advances in synthetic and chemical biology have successfully demonstrated the ability to encode diverse nonstandard amino acids (nsAAs), including physiologically relevant PTMs, into proteins. Recent advances in the development of genomically recoded organism (GROs) – recoded strains of E. coli with open coding channels – and engineered translation systems that encode PTMs (e.g., phosphoserine) have allowed activation of human phosphoproteins. These advances have defined active protein states at a molecular level, revealed substrate networks, and implicated new function for disease-relevant PTMs. However, two key challenges have emerged that preclude a thorough understanding of these protein networks and limit the translation of such insights into targeted clinical solutions. First, the precise arrangement and contributions of distinct PTMs in higher order combinations that lead to active protein states is often unknown and hard to decipher. Second, dissecting the unique combination of PTMs that govern protein-protein interactions which underlie cellular signaling and physiological function is poorly understood. Third, the development of targeted therapeutics that precisely target diseased protein states remains an active area of translational development. Specific Aims: In this proposal, we seek to utilize a recently developed next- generation GRO to develop mutually orthogonal translational machinery to simultaneously encode phosphorylation and acetylation PTMs at the UAG and UGA codons (Aim 1), utilize these dual encoding capabilities to develop a high throughput method to discover PTM-mediated intracellular protein binders (Aim 2), and engineer small protein binders to neutralize phosphorylated-oligomeric Tau proteins (Aim 3). This work will be significant because it will enable the biosynthesis of active human proteins genetically encoded with phosphorylation and acetylation moieties to dissect PTM-mediated protein-protein binding interactions. These findings can reveal complex biomolecular interactions that recapitulate human protein networks that are difficult to isolate and study in their native contexts. These technologies establish a new approach to dissect PTM biomarkers at molecular specificity and pave the way for a new synthetic biology-based drug discovery paradigm.