Protein-Specific Crowding Accelerates Aging in Protein Condensates

Macromolecular crowding agents, such as poly(ethylene glycol) (PEG), are often used to mimic cellular cytoplasm in protein assembly studies. Despite the perception that crowding agents have an inert nature, we demonstrate and quantitatively explore the diverse effects of PEG on the phase separation and maturation of protein condensates. We use two model proteins, the FG domain of Nup98 and bovine serum albumin (BSA), which represent an intrinsically disordered protein and a protein with a well-established secondary structure, respectively. PEG expedites the maturation of Nup98, enhancing denser protein packing and fortifying interactions, which hasten beta-sheet formation and subsequent droplet gelation. In contrast to BSA, PEG enhances droplet stability and limits the available solvent for protein solubilization, inducing only minimal changes in the secondary structure, pointing toward a significantly different role of the crowding agent. Strikingly, we detect almost no presence of PEG in Nup droplets, whereas PEG is moderately detectable within BSA droplets. Our findings demonstrate a nuanced interplay between crowding agents and proteins; PEG can accelerate protein maturation in liquid-liquid phase separation systems, but its partitioning and effect on protein structure in droplets is protein specific. This suggests that crowding phenomena are specific to each protein-crowding agent pair.