Thesis advisor: Abhishek Chatterjee / The study of protein structure and function has advanced significantly with the development of genetic code expansion (GCE) technology for the incorporation of noncanonical amino acids (ncAAs), revolutionizing synthetic biology by enabling the introduction of novel functionalities into proteins. Within eukaryotic systems, these advancements have paved the way for deeper investigations into complex protein functions critical to human biology and have spurred the development of innovative biotherapeutic solutions.The work described within this dissertation has aimed to further advance various applications of mammalian GCE. This includes the construction of next-generation homogenous antibody-drug conjugates (ADCs) both using a genetically encoded photocaged cysteine and with a dual incorporation system for the construction of a dual-drug conjugate. Multiple new platforms were developed for the incorporation of two or even three ncAAs within a single protein, utilizing a novel aaRS/tRNA pair and evolved hyper-efficient tRNAs. GCE-enabled precise protein modification was also utilized to spectroscopically study the conformational dynamics of dimeric EGFR. Additionally, platforms were established for the precise installation of post-translational modification (PTM) mimics within mammalian proteins, allowing for their programmed activation. Finally, an innovative strategy for the study of protein-protein interactions using genetically encoded photocrosslinkers was developed. Collectively, these efforts have contributed to the development of novel tools for studying protein function in mammalian cells and advancing the creation of new biotherapeutics through GCE technology. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.