Simultaneous Detection of Distinct Ubiquitin Chain Topologies by (19)F NMR

[Image: see text] The dynamic interplay between ubiquitin (Ub) chain construction and destruction is critical for the regulation of many cellular pathways. To understand these processes, it would be ideal to simultaneously detect different Ub chains as they are created and destroyed in the cell. This objective cannot be achieved with existing detection strategies. Here, we report on the use of (19)F Nuclear Magnetic Resonance (NMR) spectroscopy to detect and characterize conformationally distinct Ub oligomers. By exploiting the environmental sensitivity of the (19)F nucleus and the conformational diversity found among Ub chains of different linkage types, we can simultaneously resolve the (19)F NMR signals for mono-Ub and three distinct di-Ub oligomers (K6, K48, and K63) in heterogeneous mixtures. The utility of this approach is demonstrated by the ability to interrogate the selectivity of deubiquitinases with multiple Ub substrates in real time. We also demonstrate that (19)F NMR can be used to discern Ub linkages that are formed by select E3 ligases found in pathogenic bacteria. Collectively, our results assert the potential of (19)F NMR for monitoring Ub signaling in cells to reveal fundamental insights about the associated cellular pathways.