title: 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.
authors.affiliation: Department of Chemistry, University of Wisconsin-Madison , 1101 University Avenue, Madison, Wisconsin 53706, United States.
authors.forename: Sujan S
authors.initials: SS
authors.lastname: Shekhawat
authors.forename: Grace H
authors.initials: GH
authors.lastname: Pham
authors.forename: Jyothiprashanth
authors.initials: J
authors.lastname: Prabakaran
authors.forename: Eric R
authors.initials: ER
authors.lastname: Strieter
mesh_terms: D005461:Fluorine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D025801:Ubiquitin; D005461:Fluorine; D044767:Ubiquitin-Protein Ligases