Simultaneous Detection of Distinct Ubiquitin Chain Topologies by ¹⁹F NMR

Abstract

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 19F Nuclear Magnetic Resonance (NMR) spectroscopy to detect and characterize conformationally distinct Ub oligomers. By exploiting the environmental sensitivity of the 19F nucleus and the conformational diversity found among Ub chains of different linkage types, we can simultaneously resolve the 19F 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 19F 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 19F NMR for monitoring Ub signaling in cells to reveal fundamental insights about the associated cellular pathways.

SAGA DUB-Ubp8 Deubiquitylates Centromeric Histone Variant Cse4

“…The variety of ubiquitin chain versions represent not only a signal for the proteasome but also a versatile three-dimensional code, controlling many different cellular functions ( Heride et al 2014 ). There is still a great debate regarding the precise role of Ub-chain species, how their different chain length and linkage type can affect the way DUBs process them, and the meaning and translation of this ubiquitin code into biological processes ( Shekhawat et al 2014 ). The investigation of the mechanisms and the players involved represent a great challenge for the future.…”

PP2A forestalls mitophagy by dephosphorylating Parkin and ubiquitin

“…To monitor the dephosphorylation of pUb in real-time, we conjugated a trifluoromethyl probe at the K48C site of ubiquitin 33,34 , and collected a series of one-dimensional 19 F NMR spectra. As shown previously, pUb fluctuates between two conformational states, namely the relaxed state and retracted state 8,9,13 .…”

Real-time NMR monitoring of biological activities in complex physiological environments

“…Here, we highlight RT-NMR approaches that exploit the selective incorporation of 19 F fluorine to monitor protein folding and aggregation events [41], as well as ubiquitin chain assembly and disassembly, both in reconstituted ubiquitination systems and cell extracts [42]. The latter study provided insights into the activities of cellular ubiquitin ligases and deubiquitinases by delineating their site specificities at different substrate lysine residues.…”