A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro

Raymond-Smiedy, Peter; Bucknor, Barrington; Yang, Yiran; Zheng, Tongyin; Castañeda, Carlos

doi:10.1007/978-1-0716-2597-2_32

Cited by 4 publications

(7 citation statements)

References 33 publications

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“…The UBQLN2 purification protocol here is adapted from. 35 For Ub, perchloric acid was added to the lysate to a final pH of about 1. Precipitated proteins were cleared by centrifugation.…”

Section: ■ Methodsmentioning

confidence: 99%

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation

et al. 2023

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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.

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“…The UBQLN2 purification protocol here is adapted from. 35 For Ub, perchloric acid was added to the lysate to a final pH of about 1. Precipitated proteins were cleared by centrifugation.…”

Section: ■ Methodsmentioning

confidence: 99%

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation

et al. 2023

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“…Full-length UBQLN2 and UBQLN2 450-624 were expressed and purified as described previously (Raymond-Smiedy et al , 2023; Dao et al , 2018). Briefly, the constructs were transformed into Rosetta 2 (DE3) pLysS E. coli cells and were grown in LB media with a final concentration of 50 mg/L kanamycin and 34 mg/L chloramphenicol to OD 600 of 0.6-0.8, induced with 0.5 mM IPTG, and expressed overnight at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…Turbidity assays were generally performed as described in (Raymond-Smiedy et al , 2023). Protein samples were prepared by mixing different ratios of UBQLN2 450C (or full length UBQLN2) and Ub mutants/HT6-Ub/M1-Ub4 constructs (the initial concentrations of the protein stocks were doubled compared to the sample concentrations) in cold 20 mM sodium phosphate buffer pH 6.8 with 0.5 mM EDTA and 0.02 % NaN 3 .…”

Section: Methodsmentioning

confidence: 99%

“…The phase boundary for UBQLN2 LCST (lower critical solution temperature) phase transitions at specific concentrations of ligand and protein were determined from turbidity assays as described above (Raymond-Smiedy et al , 2023). The ligand hub concentrations were chosen to cover as wide a range as possible.…”

Section: Methodsmentioning

confidence: 99%

“…above(Raymond-Smiedy et al, 2023). The ligand hub concentrations were chosen to cover aswide a range as possible.…”

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confidence: 99%

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Decoding optimal ligand design for multicomponent condensates

Galagedera

Dao

Enos

et al. 2023

Preprint

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Biomolecular condensates form via multivalent interactions among key macromolecules and are regulated through ligand binding and/or post-translational modifications. One such modification is ubiquitination, the covalent addition of ubiquitin (Ub) or polyubiquitin chains to target macromolecules for various cellular processes. Specific interactions between polyubiquitin chains of different linkages and partner proteins, including hHR23B, NEMO, and UBQLN2, regulate condensate assembly or disassembly. Here, we used a library of designed polyubiquitin hubs and UBQLN2 as model systems for determining the driving forces of ligand-mediated phase transitions. Systematic decreases to the binding affinity between Ub and UBQLN2 or deviations from the optimal spacing between Ub units reduce the ability of hubs to modulate UBQLN2 phase behavior. Using an analytical model that accurately described the effects of different hubs on UBQLN2 phase diagrams, we determined that introduction of Ub to UBQLN2 condensates incurs a significant inclusion energetic penalty. This penalty competes with the hub's ability to scaffold multiple UBQLN2 molecules, thereby cooperatively amplifying phase separation. Importantly, there exists an optimal polyubiquitin hub design that maximally promotes phase separation. Hubs where Ub units are too close or too far apart inhibit phase separation. These effects on phase diagrams are encoded in the spacings between Ub units as found for naturally-occurring chains of different linkages and designed chains of different architectures, thus illustrating how the ubiquitin code regulates functionality via the emergent properties of the condensate. We expect our findings to extend to other condensates necessitating the consideration of ligand properties, including concentration, valency, affinity, and spacing between binding sites in studies and designs of condensates.

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

Robb

Dao

Ujma

et al. 2022

Preprint

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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.

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A Spectrophotometric Turbidity Assay to Study Liquid-Liquid Phase Separation of UBQLN2 In Vitro

Cited by 4 publications

References 33 publications

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation

Ion Mobility Mass Spectrometry Unveils Global Protein Conformations in Response to Conditions that Promote and Reverse Liquid–Liquid Phase Separation

Decoding optimal ligand design for multicomponent condensates

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

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