PEG 1000 as a molecular switch: Concentration-dependent aggregation and refolding in a non-aggregation-prone β-barrel protein.

Macromolecular crowding significantly influences protein folding, stability, and aggregation within cellular environments. Here, Cellular Retinoic Acid Binding Protein I (CRABP I), a highly stable β-barrel protein with low intrinsic aggregation propensity, was employed to investigate the concentration-dependent effects of PEG 1000 on temperature-assisted refolding. Structural, functional, and biological consequences of refolding were examined using spectroscopic, microscopic, rheological, ligand-binding, zeta potential, and in vivo analyses. Refolding in the absence of PEG produced soluble but structurally heterogeneous intermediates. In contrast, low PEG concentration promoted intermolecular protein-protein interactions, leading to amorphous aggregation, altered structural properties, reduced retinoic acid-binding activity, and enhanced oxidative stress and cellular damage in Drosophila melanogaster. Higher PEG concentrations suppressed aggregation and favoured structurally stabilized conformations with preserved ligand-binding function. These findings demonstrate that PEG 1000 acts as a concentration-dependent molecular switch governing the balance between aggregation and productive refolding of CRABP I.