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

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Robb, Christina Glen and Dao, Thuy P. and Ujma, Jakub and Castañeda, Carlos A. and Beveridge, Rebecca (2023) Ion mobility mass spectrometry unveils global protein conformations in response to conditions that promote and reverse liquid-liquid phase separation. Journal of the American Chemical Society, 145 (23). pp. 12541-12549. ISSN 1520-5126 (https://doi.org/10.1021/jacs.3c00756)

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Abstract

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.

ORCID iDs

Robb, Christina Glen, Dao, Thuy P., Ujma, Jakub, Castañeda, Carlos A. and Beveridge, Rebecca ORCID logoORCID: https://orcid.org/0000-0003-0320-6496;
CORE (COnnecting REpositories)