A new Förster resonance energy transfer-based platform to track substrate ubiquitination by fluorescence

Post-translational modification of protein by ubiquitin (Ub) alters the stability, subcellular location, or function of the target protein, thereby impacting numerous biological processes and directly contributing to myriad cellular defects or disease states, such as cancer. Tracking substrate ubiquitination by fluorescence provides opportunities for advanced reaction dynamics studies and for translational research including drug discovery. However, fluorescence based techniques in ubiquitination studies remain underexplored at least partly due to challenges associated with Ub chain complexity and requirement for additional substrate modification. Here we describe a general strategy, Förster resonance energy transfer (FRET) di-ubiquitination, to track substrate ubiquitination by fluorescence.This platform produces a uniform di-Ub product depending on specific interactions between a substrate and its cognate E3 Ub ligase. The di-ubiquitination creates proximity between the Ub-linked donor and acceptor fluorophores, respectively, enabling energy transfer to yield a distinct fluorescent signal. FRET di-ubiquitination relies on Ub-substrate fusion, which can be implemented using either one of the two validated strategies. Method one is the use of recombinant substrate-Ub fusion, applicable to all substrate peptides that can bind to E3. Method two is a chemo-enzymatic ligation approach that employs synthetic chemistry to fuse Ub with a substrate peptide containing desired modification. Taken together, our new FRET-based di-ubiquitination system provides a timely technology of potential to advance both basic research and translation sciences.