Modulatory Effect of the Polyphenol EGCG toward the Fibrillation of Monomers and Preformed Aggregates of α-Synuclein in the Presence of Small-Molecule and Polymeric Crowders

Epigallocatechin gallate (EGCG) has been widely studied for its inhibitory effects on amyloidogenic protein aggregation, yet its effect on the intrinsically disordered neuronal protein α-synuclein (α-syn), which is implicated in many neurodegenerative disorders, remains unclear, especially under molecular crowding conditions that mimic cellular environments. However, recent studies have reported contrasting effects of EGCG on α-syn aggregation. This study comprehensively examines the effect of EGCG on monomeric α-syn aggregation and preformed aggregates in the absence and presence of small (polyol osmolytes) and large (PEG-8000) molecular crowders. Our results show that EGCG delays the lag phase of α-syn aggregation in the presence of polyols, whereas no significant change is observed in the presence of the large molecular weight crowder PEG8K. Atomic force microscopy images reveal that EGCG distinctly redirects α-syn to form oligomers and amorphous aggregates under crowded conditions. Furthermore, while α-syn, in the presence of a combination of EGCG and PEG8K, exhibits random coil and α-helical conformations, it shows a completely disordered conformation in its absence. Interestingly, the addition of EGCG to preformed aggregates does not alter aggregate size but impacts their morphology significantly, as shown by light scattering and AFM. Despite the changes in the morphology of aggregates, a predominantly β-sheet-rich secondary structure was observed by CD spectroscopy after EGCG addition, suggesting complexity in the action of EGCG under crowding conditions. Our study highlights the critical role played by the physicochemical nature of the cellular environment, consisting of small-molecule osmolytes as well as macromolecular crowders, in protein aggregation and its modulation by small-molecule ligands such as EGCG.