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Optimizing a biosensor‐based assay for ubiquitination activation

Ubiquitination is a post‐translational modification which regulates protein degradation, DNA damage signaling, and inflammatory response. Three transfer enzymes catalyze the reaction’s pathway: E1 activating enzyme, E2 conjugating enzyme, and E3 ligating enzyme, respectively. Despite the identification of these enzymes’ structure and function, a complete understanding of their catalytic mechanisms remains unclear. Clarification of the ubiquitination mechanism could serve medicinal strategies due ubiquitin’s (Ub’s) involvement with target protein degradation. Existing assays for ubiquitination are either too imprecise, expensive, or radioactive for common usage, thus we are trying to develop a cheap, continuous assay which can make ubiquitination research more accessible. Our approach depends on the detection of ATP as E1 cleaves ATP into pyrophosphate (PPi) and AMP for Ub‐activation. Previous fluorescent assay designs for Ub‐E1 activation were attempted and examined including a terbium‐norfloxacin assay and a chloroacetaldehyde assay, but each showed limitations. The terbium‐norfloxacin assay detected ATP fluorescence, but interfered with the protein pathway, while the chloroacetaldehyde assay used organic sensors to modify ATP, but with little success. We have since turned to adapting a genetically‐encoded nucleotide biosensor, Perceval, toward measuring ATP hydrolysis of E1 in the ubiquitination pathway. Developed in the Yellen Lab at Harvard Medical School, Perceval was designed to report real‐time changes in cellular energy as a result of its competitive binding of ATP and ADP. We have worked to tailor the Perceval model towards measuring solely the ATP hydrolysis of ubiquitination in vitro, starting with E1 activation. After optimizing Perceval’s purification, we showed Perceval has a high affinity for ATP without the ADP competitive binding aspect, and ensured AMP and PPi do not interfere with the biosensor. We similarly tested solutions containing different Ub protein substrates and E1s, and found that Perceval’s affinity for ATP is not interrupted by critical components of the ubiquitination pathway. When directly applied to ubiquitination, fluorescence readings were taken at single‐time points in the ubiquitination pathway and over a continuous duration. Preliminary data suggest Perceval could be a potential biosensor for measuring Ub activation in real time. Future directions will look to further optimize assay conditions as well as test other biosensors for comparison.