PEG-mCherry interactions beyond classical macromolecular crowding

The dense cellular environment influences bio-macromolecular structure, dynamics, interactions and function. Despite advancements in understanding protein-crowder interactions, predicting their precise effects on protein structure and function remains challenging. Here, we elucidate the effects of PEG-induced crowding on the fluorescent protein mCherry using molecular dynamics simulations and fluorescence-based experiments. We identify and characterize specific PEG-induced structural and dynamical changes in mCherry. Importantly, we find interactions in which PEG molecules wrap around specific surface-exposed residues in a binding mode previously observed in protein crystal structures. Fluorescence correlation spectroscopy experiments capture PEG-induced changes, including aggregation, suggesting a potential role for the specific PEG-mCherry interactions identified in simulations. Additionally, mCherry fluorescence lifetimes are influenced by PEG and not by the bulkier crowder dextran, highlighting the importance of crowder-protein soft interactions. This work augments our understanding of macromolecular crowding effects on protein structure and dynamics. ### Competing Interest Statement The authors have declared no competing interest.