Excited to share our new paper! It was a really enjoyable collaboration with the Keating Lab.
onlinelibrary.wiley.com/doi/full/10....
We asked a simple question:
Do all crowded environments affect protein phase separation the same way?
Short answer: no.
This was a really fun rewarding collaboration! Huge thanks to:
Chris Keating for her insight and guidance
Jake Shaffer for the confocal imaging and collaboration
Scott Showalter for his support and mentorship
And thank you to the reviewers for their constructive feedback that improved this paper.
More broadly, this work shows that we can use NMR to map crowding effects residue by residue and start building more realistic models for how crowding influences phase separation.
The take-home message is that - not all crowding is equal.
Different crowders create different microscopic environments, and these differences matter for protein phase behavior.
Even when environments look similar at the residue level (PEG, lysozyme, E. coli cytosol), the magnitude of effects differs, and this leads to very different outcomes:
droplets vs. no droplets / aggregates.
One interesting case is dextran.
Even though it promotes phase separation like other polymer crowders, its NMR signature is very different from them and from the self-reference.
This suggests different underlying mechanisms, even among polymer crowders.
We found that all crowders increase self-interactions to some extent, but they also create distinct chemical environments.
Crowders don’t just occupy space. They also interact differently with the protein system.
Under crowding conditions, peaks also shift, but differently depending on the crowder.
We compared relative residue-level patterns of these shifts across conditions and asked:
Are crowders simply enhancing self-interactions, or doing something else?
Even without crowders, increasing protein concentration shifts NMR peaks. We interpret this as increased self-interactions.
This becomes our “reference” for comparison.
We used carbon direct-detect NMR (our favorite). This lets us easily see prolines, which are highly abundant in the CTD and other IDRs and not very straightforward to see in standard NMR.
So we get a much more complete picture of the protein.
We then used NMR to zoom in at the residue level.
The CTD has repeating YSPTSPS units, which makes it a great system to ask:
How does each residue “sense” different environments?
At the macroscopic level, polymer crowders consistently promote droplet formation, while protein crowders and E. coli cytosol do not and can even cause aggregation.
So already: phase separation depends on crowder identity.
We looked at phase separation of the Pol II CTD (an IDR) under different crowding conditions:
- Polymer crowders (PEG, dextran, Ficoll)
- Protein crowders (BSA, lysozyme)
- Reconstituted E. coli lysate
They do not behave the same.
Excited to share our new paper! It was a really enjoyable collaboration with the Keating Lab.
onlinelibrary.wiley.com/doi/full/10....
We asked a simple question:
Do all crowded environments affect protein phase separation the same way?
Short answer: no.
Now published! Phosphorylation patterns modulate transient secondary structures of the intrinsically disordered CTD of RNA polymerase II www.sciencedirect.com/science/arti...
Excited to share our new review on recent conceptual breakthroughs in IDPs with the help of NMR! From molecular descriptions to cellular functions of intrinsically disordered protein regions
pubs.aip.org/aip/bpr/arti...