Liquid–liquid phase separation (LLPS) is a process
by which
biomacromolecules, particularly proteins, condense into a dense phase
that resembles liquid droplets. Dysregulation of LLPS is implicated
in disease, yet the relationship between protein conformational changes
and LLPS remains difficult to discern. This is due to the high flexibility
and disordered nature of many proteins that phase separate under physiological
conditions and their tendency to oligomerize. Here, we demonstrate
that ion mobility mass spectrometry (IM–MS) overcomes these
limitations. We used IM–MS to investigate the conformational
states of full-length ubiquilin-2 (UBQLN2) protein, LLPS of which
is driven by high-salt concentration and reversed by noncovalent interactions
with ubiquitin (Ub). IM–MS revealed that UBQLN2 exists as a
mixture of monomers and dimers and that increasing salt concentration
causes the UBQLN2 dimers to undergo a subtle shift toward extended
conformations. 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 correlated
with UBQLN2’s ability to phase separate. Overall, delineating
protein conformations that are implicit in LLPS will greatly increase
understanding of the phase separation process, both in normal cell
physiology and disease states.