Polyethylene Glycol-Mediated Crowding: Insights through Protein Dynamics and Phase Separation

Polyethylene glycol (PEG) is widely recognized as a versatile model system for investigating protein-polymer interactions owing to its tunable physicochemical properties and hence has also been commonly used as macromolecular crowding agents. PEGs exist spanning a range of molecular weights, thereby exhibiting a diverse set of interactions with biomolecules. However, a systematic comparison between the different PEGs with regard to their properties as macromolecular crowders is hard to come by. The present work adopts a comprehensive approach in which both the behavior of a protein and the intrinsic characteristics of a few of the PEG-based crowding agents have been investigated. The effect of different PEGs (PEG 1, PEG 4, PEG 8, PEG 10, and PEG 20) on the global (secondary structure) and pseudoglobal (relative domain displacement) conformations and local dynamics of the enzyme AK3L1 (an isoform of adenylate kinase) has been studied using circular dichroism, FRET, and solvation, respectively. The local dynamics results are consistent with FRET observations, indicating that excluded-volume effects induce structural compaction of AK3L1 while simultaneously increasing the rigidity in the immediate microenvironment comprising the protein matrix and hydration shell around the Cys-132 residue in the LID domain. Among all PEGs, PEG 10 induces the strongest retardation of solvation dynamics, a conclusion further supported by diffusion measurements of TMR-labeled AK3L1. To gain deeper insights into the crowded milieu, diffusion measurements of fluorescently labeled crowders and proteins (TMR-AK3L1 and TMR-BSA) were performed using fluorescence correlation spectroscopy, with nanoparticle tracking analysis serving as a complementary technique. Together, these approaches enabled the quantitative characterization of diffusion behavior and the emergence of crowder-induced self-assemblies across a broad range of PEG concentrations. Additionally, we investigated and systematically compared PEG-induced phase separation in binary mixtures with other macromolecular crowders, namely, Ficoll and dextran, using confocal microscopy and fluorescence recovery after photobleaching. Our results reveal that dextran-PEG mixtures exhibit a substantially higher degree of incompatibility and more pronounced phase separation in comparison to Ficoll-PEG systems. Notably, the extent of phase separation increases with increasing molecular weight of PEG, underscoring the critical role of crowder size in governing the phase behavior. Taken together, our results provide detailed insights that stand to advance our understanding of how PEG-based crowders influence enzyme dynamics, along with a better comprehension of the underlying differences in the solution properties of the PEGs, and more importantly, the biophysical basis of condensate formation.