Ion mobility mass spectrometry unveils global protein conformations in response to conditions that promote and reverse liquid-liquid phase separation

Liquid-liquid phase separation (LLPS) is a process by which proteins and macromolecules condense into a dense phase that resembles liquid droplets. Ubiquilin-2 (UBQLN2), a ubiquitin (Ub)-binding protein with intrinsically disordered regions, undergoes LLPS alone under physiological conditions and colocalises with stress granules, which are a type of membraneless organelle hypothesised to form via phase separation. LLPS of UBQLN2 is driven by high salt concentration and reversed by the presence of Ub. However, the effects that these conditions have on the overall conformation of UBQLN2 remain unknown. Using ion mobility mass spectrometry (IM-MS), we discovered that UBQLN2 exists as a mixture of monomers and dimers, and that increasing salt concentration causes the UBQLN2 dimers to undergo a subtle shift towards extended conformations. In the presence of Ub, we observed 2:1 and 2:2 UBQLN2:Ub complexes which have compact geometries compared to free UBQLN2 dimers. Together, these data suggest that extended conformations of UBQLN2 are correlated with the ability of UBQLN2 to phase separate. Overall, delineating the conformations that are implicit in LLPS will greatly increase understanding of the process, both in normal cell physiology and in disease states such as in amyotrophic lateral sclerosis that can be caused by mutations to UBQLN2. This work demonstrates the strength of IM-MS in uncovering the molecular mechanisms of LLPS using full-length protein constructs.