Thesis
An investigation into the conformational dynamics of ubiquilin-2 and its interactions with ubiquitin and polyubiquitin by native ion mobility mass spectrometry
- Creator
- Rights statement
- Awarding institution
- University of Strathclyde
- Date of award
- 2026
- Thesis identifier
- T18084
- Person Identifier (Local)
- 202159253
- Qualification Level
- Qualification Name
- Department, School or Faculty
- Abstract
- Ubiquilin-2 (UBQLN2) is an intrinsically disordered, ubiquitin (Ub) shuttle protein. Its function in cells is to deliver ubiquitinated substrates to the appropriate degradation machinery. It is effective in doing so, due to the Ub-like domain and Ub-associating domain architecture. A mechanism central to UBQLN2’s function is its ability to undergo phase separation, due to its intrinsic disorder. Phase separation of UBQLN2 is promoted by an increase in salt concentration and disrupted by addition of monoUb. PolyUb chains also modulate phase separation, with different responses depending on chain length and Ub linkage type. Removal of key UBQLN2 domains also results in altered phase separation activities. Prior to this work, no structural information was available for full length UBQLN2, neither alone nor in complex with Ub/polyUb. This work explores the conformational preferences of UBQLN2 with native ion-mobility mass spectrometry. This technique is ideally positioned to measure phase separating, intrinsically disordered proteins as conformations which form en route to phase separation can be measured while the protein is still in its soluble form. First, native ion-mobility mass spectroscopy was used to measure UBQLN2 when phase separation is promoted and disrupted, by an increase in salt concentration and addition of monoUb. This work revealed that UBQLN2 is highly dynamic in solution and elongates at high salt and compact conformations are stabilised upon addition of monoUb. Next, UBQLN2 was measured in complex with K48- and K63-linked PolyUb chains and the chains were measured alone. It was observed that PolyUb chains behave differently in the gas phase than solution phase and molecular mechanisms were proposed for their recognition by UBQLN2. Lastly, we delineate the role of each domain on the conformational ensemble and oligomeric state of UBQLN2. We demonstrate that there is a balance of interactions between domains which stabilises compact UBQLN2 conformations and identify the domain responsible for UBQLN2 oligomerisation. This thesis has provided unique insight into UBQLN2 and its function as a ubiquitin shuttle protein and has strengthened native ion-mobility mass spectrometry as a biophysical technique to study phase separating, intrinsically disordered protein systems.
- Advisor / supervisor
- Beveridge, Rebecca
- Resource Type
- DOI
- Embargo Note
Relations
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File | 2026-09-22 | University of Strathclyde |