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.
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.
There is mounting evidence that crystal nucleation from supersaturated solution involves the formation and reor-ganisation of pre-nucleation clusters contradicting classical nucleation theory. Here, a wide range of amino acids and peptides is investigated using light scattering, mass spectrometry, and in-situ terahertz Raman spectroscopy to demonstrate that the presence of amorphous aggregates with a wide range of sizes is a general phenomenon in supersaturated solutions. Additionally, these amorphous aggregates act as intermediates for laser-induced crystal nucleation. These observations are inconsistent not only with classical nucleation theory, but also non-classical theories involving liquid-liquid phase separation, requiring a new approach.
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