khushi singh.in

Research · Completed · May–July 2024

Droplet or clump? Phase separation of the prion protein

IISER Pune · Prof. Jayant B. Udgaonkar

Proteins from: Mus musculus

The question

Under which conditions of salt, pH and concentration does the mouse prion protein form liquid-like droplets or aggregates?

Many proteins undergo liquid–liquid phase separation (LLPS), forming dense droplets without a membrane. For the prion protein, the conditions that favour droplets versus aggregates matter because misfolded prion protein causes neurodegenerative prion diseases. I varied salt, pH and protein concentration and followed phase behaviour with UV–visible spectrophotometry.

The basics: droplets inside cells

Why ask this?

Proteins are not always dissolved evenly. Many can phase-separate into dense, liquid-like droplets, much as oil separates from vinegar. Established For the prion protein this matters: when it misfolds and aggregates, it can template its bad shape onto other copies, causing rare but devastating brain diseases. Established

Whether the protein forms reversible droplets or harmful clumps depends on its surroundings.

From droplets to solids

Phase separation starts when a protein’s concentration and its surroundings cross a phase boundary: the solution splits into a dense, protein-rich phase and a dilute one (Banani et al. 2017; Alberti et al. 2019). Liquid droplets are round, fuse and exchange molecules with their surroundings; with time, some harden into gels or solids (Patel et al. 2015). Established

The prion protein has a long disordered N-terminal region and a folded C-terminal domain. It forms a liquid phase in vitro (Kostylev et al. 2018). Its droplets can age into a β-sheet-rich, proteinase-resistant gel, which needed the N-terminal region (amino acids 23–89) and kosmotropic (water-structuring) salts (Tange et al. 2021). Emerging Phase separation also changes the shape of the N-terminal region itself (Pal & Udgaonkar 2026). Emerging

0:20 / 0:26

Ageing: liquid to solid. With time, prion protein droplets can harden into a gel and then a β-sheet-rich, proteinase-resistant solid. This needed the protein’s disordered N-terminal region and kosmotropic salts (Tange et al. 2021).

Droplets scatter light, so turbidity is a simple readout of phase separation. That is why I followed it by UV–visible spectrophotometry.

What I did

  • I changed salt, pH and protein concentration systematically.
  • I followed droplet formation and aggregation by UV–visible spectrophotometry (turbidity).
  • I read into protein misfolding and its links to neurodegeneration.

What I took away

Physics and chemistry decide biology’s states. Small changes in conditions can tip a protein from a healthy droplet into a harmful clump. The basics are in droplets inside cells.

Why it matters

Understanding how condensates age or solidify is an active frontier in neurodegeneration research. Emerging

Methods used

  • Protein biochemistry
  • UV–visible spectrophotometry (turbidity)
  • Varying solution conditions (salt, pH, concentration)

What these methods are →

References

  1. Pal S, Udgaonkar JB (2026) Phase separation leads to significant conformational change in the N-terminal region of the prion protein. Protein Science 35:e70742. doi:10.1002/pro.70742
  2. Banani SF, Lee HO, Hyman AA, Rosen MK (2017) Biomolecular condensates: organizers of cellular biochemistry. Nature Reviews Molecular Cell Biology 18:285–298. doi:10.1038/nrm.2017.7
  3. Alberti S, Gladfelter A, Mittag T (2019) Considerations and challenges in studying liquid–liquid phase separation and biomolecular condensates. Cell 176:419–434. doi:10.1016/j.cell.2018.12.035
  4. Patel A, Lee HO, Jawerth L, et al., Alberti S (2015) A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation. Cell 162:1066–1077. doi:10.1016/j.cell.2015.07.047
  5. Kostylev MA, Tuttle MD, Lee S, et al., Strittmatter SM (2018) Liquid and hydrogel phases of PrPC linked to conformation shifts and triggered by Alzheimer’s amyloid-β oligomers. Molecular Cell 72:426–443. doi:10.1016/j.molcel.2018.10.009
  6. Tange H, Ishibashi D, Nakagaki T, et al., Nishida N (2021) Liquid–liquid phase separation of full-length prion protein initiates conformational conversion in vitro. Journal of Biological Chemistry 296:100367. doi:10.1016/j.jbc.2021.100367