Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid-Liquid Phase Separation

摘要

Liquid-liquid phase separation (LLPS) is a processby whichbiomacromolecules, particularly proteins, condense into a dense phasethat resembles liquid droplets. Dysregulation of LLPS is implicatedin disease, yet the relationship between protein conformational changesand LLPS remains difficult to discern. This is due to the high flexibilityand disordered nature of many proteins that phase separate under physiologicalconditions and their tendency to oligomerize. Here, we demonstratethat ion mobility mass spectrometry (IM-MS) overcomes theselimitations. We used IM-MS to investigate the conformationalstates of full-length ubiquilin-2 (UBQLN2) protein, LLPS of whichis driven by high-salt concentration and reversed by noncovalent interactionswith ubiquitin (Ub). IM-MS revealed that UBQLN2 exists as amixture of monomers and dimers and that increasing salt concentrationcauses the UBQLN2 dimers to undergo a subtle shift toward extendedconformations. UBQLN2 binds to Ub in 2:1 and 2:2 UBQLN2/Ub complexes,which have compact geometries compared to free UBQLN2 dimers. Together,these results suggest that extended conformations of UBQLN2 are correlatedwith UBQLN2's ability to phase separate. Overall, delineatingprotein conformations that are implicit in LLPS will greatly increaseunderstanding of the phase separation process, both in normal cellphysiology and disease states.