Date of Award

Fall 10-1-2021

Document Type


Degree Name

Doctor of Philosophy (PhD)


Cellular and Molecular Physiology

First Advisor

Rinehart, Jesse


Protein phosphorylation is a ubiquitous post-translational modification that governs signaling cascades and protein-protein interactions. Orthogonal translation systems, in which an orthogonal aminoacyl-tRNA synthetase and tRNA pair have been repurposed for insertion of a non-standard amino acid, have been developed for co-translational insertion of phospho-amino acids. Here, we systematically developed variants of phosphoserine and phosphothreonine orthogonal translation systems to minimize cellular toxicity and maximize translational fidelity. We characterized novel tRNA constructs to enhance cellular fitness and change decoding functionality. Multiple genetic background strains were developed to enhance the incorporation of phospho-amino acids into recombinant proteins. Utilizing large-scale DNA synthesis, we were able to produce serine, threonine, and tyrosine libraries representative of the entire known human phosphoproteome. By pairing our libraries with a bimolecular fluorescence complementation assay, we were able to characterize novel phosphorylation-dependent protein-protein interactions for phosphoserine, phosphothreonine, and phosphotyrosine. Genetically encoded phosphothreonine enabled the discovery of a new activation mechanism for the protein kinase CHK2 and proteome-wide surveys of its target substrates. Finally, we developed Hi-P+ to couple kinase substrate discovery with phosphorylation-dependent protein-protein interactions. This work enables kinase-specific, proteome-wide surveys of multi-level phosphorylation-dependent interactions with phosphosite resolution.