Thesis advisor: Abhishek Chatterjee / Förster Resonance Energy Transfer (FRET) offers a powerful approach to study biomolecular dynamics in vitro as well as in vivo. The ability to apply FRET imaging to proteins in living cells provides an excellent tool to monitor important dynamic events such as protein conformational changes, protein-protein interactions, and proteolysis reactions. However, selectively incorporating two distinct fluorophores into the target protein(s) that are capable of FRET interaction within the complex cellular milieu is challenging. Consequently, terminal fusion to genetically encoded fluorescent proteins has emerged as the predominant labeling strategy for FRET studies in vivo. However, a major limitation of this strategy stems from the large size of the fluorescent proteins, which may perturb the native properties of the target, and restricted attachment only to the termini of the target. We reasoned that using genetically encoded fluorescent unnatural amino acids would overcome several of these challenges associated with currently available labeling strategies owing to their small size and the ability to introduce them site- specifically and co-translationally. Here, we report the use of the fluorescent unnatural amino acid “Anap” as a FRET donor with green and yellow fluorescent protein acceptors. We demonstrate the utility of this labeling strategy using proteolysis and conformational change models, and step towards in vivo studies by further developing a proteolysis system in cell lysates. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.