Residue-level mapping of crowding effects on protein phase separation

Protein liquid–liquid phase separation has emerged as a key mechanism in cellular organization. While the crowded environment inside cells is expected to influence this process, how crowding shapes the chemical environment and impacts protein phase separation remains largely unknown. Here, we use NMR spectroscopy to map residue-level crowding effects on the intrinsically disordered region of RNA polymerase II under different conditions, including polymer- and protein-based crowders, as well as 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, our results provide a strategy to evaluate their chemical influence and to design more physiologically relevant in vitro crowding models. More broadly, this framework enables systematic probing of residue-level influences in complex, cell-like environments.