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bioRxiv · 10.1101/2025.10.02.680141

Residue-level mapping of crowding effects on protein phase separation

Chen, W.·Shaffer, J. M.·Keating, C. D.·Showalter, S. A.
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Abstract

Proteins can undergo liquid-liquid phase separation to form condensates within the crowded cellular environment. In vitro, polymer-based crowding agents are widely used to mimic this effect, but how their chemical environments influence phase separation compared to native cytosol remains unclear. Here, we use NMR to probe the chemical influences on the intrinsically disordered region of RNA polymerase II under different crowding conditions, including polymer-based crowders, protein-based crowders, and reconstituted E. coli cytosol. We find a general trend of enhanced protein self-interactions across all conditions, but also distinct chemical environments that depend on crowder identity, reflecting changes in preferential interactions. Given the widespread use of polymer crowders in phase separation studies, our results establish a strategy to dissect the microscopic role of crowders and lay the foundation for designing more physiologically relevant in vitro crowding models of protein phase separation. More broadly, this framework enables systematic probing of residue-level environmental influences in complex settings including within the cellular milieu.

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Chen, W., Shaffer, J. M., Keating, C. D., Showalter, S. A.. 2025-10-04. Residue-level mapping of crowding effects on protein phase separation. https://doi.org/10.1101/2025.10.02.680141

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