Development of a synthetic biology tool to engineer proteins with multiple modifications and elucidate their binding function

Human signaling networks involve large sets of proteins exhibiting highly complex patterns of posttranslational modifications (PTMs) that govern their structure and function. While tremendous progress has been made in identifying PTMs and elucidating the organizational principles of cellular proteins1 (e.g., protein interaction Protein interactions have had a transformative impact on modern medicine. Precision biologics have resulted in breakthroughs in treating diseases (e.g., cancer) and motivate efforts to develop molecular probes that reveal how PTMs could define new targets for targeted drugs. An underlying problem is that protein complexes differentiated by PTMs cannot be separated from non-modified complexes in mammalian cell-based studies. Thus, our knowledge of how PTMs dictate organizational principles of all cellular proteins in human cells is fragmented and few protein domains able to differentiate complex PTM structure have been identified. Here, we aim to leverage advances in protein chemistry and protein engineering to develop an integrated genome and protein engineering high-throughput tool that can differentiate specific PPIs governed by multiple PTMs. Specifically, we have engineered a novel synthetic biology-based platform – genomically recoded organisms (GROs) – which possess open codons that allow two distinct PTMs to be placed in proteins individually or in combinations with precision. We have used this tool to develop Hi-P, a protein engineering platform where phosphorylation sites and phosphoprotein binding are genetically encoded allowing their interactions to be systematically defined. Here we leverage a strong foundation of expertise and preliminary data to support the development of an enabling GRO tool to elucidate how PTMs influence the organization and function of cells. In Aim 1 we will use our new GRO to develop mutually orthogonal translational machinery to simultaneously encode two PTMs, phosphotyrosine and acetylation, at the UAG and UGA codons. In Aim 2 we will develop a dual PTM HiP platform allowing PDBs that recognize two distinct post-translational modifications to be identified in genome-wide screens. This work will be innovative as protein network interactions are organized by diverse combinations of PTMs that have long been inaccessible to researchers. Our work, centered on combined genome and protein translation engineering, provides access to PTMs and removes this bottleneck. Project Number: 1R01GM163196-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Jesse Rinehart | Institution: YALE UNIVERSITY, NEW HAVEN, CT | Award Amount: $2,014,434 | Activity Code: R01 | Study Section: Chemical Biology and Probes Study Section[CBP] View on NIH RePORTER: https://reporter.nih.gov/project-details/11274635