Ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) are proteins that perform post-translational modifications on protein substrates which promote essential functions such as inflammatory response, DNA repair, and protein degradation. Three transfer enzymes catalyze the ubiquitination pathway: E1, E2, and E3 which activate, conjugate, and ligate the Ub to its target protein, respectively. Despite the identification of these enzymes’ structure and function, a complete understanding of their catalytic mechanisms remains unclear. Elucidation of the ubiquitination mechanism could serve strategies for cancer treatment and other medicinal targets due to the degradation and activity-altering aspect of Ubls towards deregulated or misfolded proteins. Available assays for ubiquitination include gel-based assays, which do not give quantitative data in real time, the luciferase assay which can become costly, as well as assays that use radiotracers. We are trying to develop a continuous, relatively cheap, non-radioactive assay which can make ubiquitination research more accessible. Our approach uses fluorescent biosensors to monitor ATP hydrolysis of E1 in the ubiquitination pathway in order to measure enzyme activity. The ultimate goal is to couple this reaction to E2 and E3 to apply the assay to the entire ubiquitination pathway. The genetically encoded biosensor iATPSnFr1.0, also unofficially known as Bohemian, was developed for ATP fluorescence detection in vivo. We chose to tailor the “Bohemian model” towards measuring ATP hydrolysis of ubiquitination in vitro. To better understand the mechanism of fluorescence and influence of structure on activity, we have applied Bohemian to kinetic assay titrations and techniques for structural characterization including Small Angle X-ray Scattering (SAXS) and computational modeling. After optimizing Bohemian's purification, we showed Bohemian has a high affinity for ATP. We have also developed the first and only structural model of this protein. SAXS data collected on the Bohemian protein has suggested dimerization of the biosensor, which disagrees with the original intentions of the biosensor design indicated in primary literature. Future directions will look to investigate optimal assay conditions for measuring kinetics of ubiquitination and using our structural model to improve the biosensor.