Biophysical and computational insights into glycol-mediated modulation of amyloid dye affinity.

Protein aggregation is main pathological hallmark of numerous neurodegenerative disorders. The characterization of protein aggregates is critical for understanding their role in disease progression as well as for developing therapeutic interventions. Thus, to study protein folding and aggregation, molecular crowding agents commonly being used to mimic the in vivo condition. Moreover, histological dyes such as Thioflavin T (ThT), 8-Anilino-1-naphthalenesulfonic acid (ANS), and Congo Red (CR) are prominent amyloid-sensitive dyes, each exhibiting distinct binding and fluorescence characteristics upon binding to amyloid fibrils. In this study, we investigated the effect of macromolecular crowding agents like Ethylene glycol (EG) and Polyethylene glycol (PEG) on binding and fluorescence properties of histological dyes. Various spectroscopic techniques revealed that there was increase and shift in the fluorescence of ThT and ANS with increasing concentration and molecular weight of PEGs and EG. Additionally, CR absorbance spectra showed structural changes in the dyes upon interaction with glycols. Further, microscopy showed altered morphology of dyes in the presence of these compounds. Moreover, molecular docking studies show that both PEGs and EG interact with the dye, thereby indicating that PEGs and EG potentially modulate the binding behavior of dyes to the protein aggregates. This study reveals that amyloid-sensitive dyes show non-specific binding with crowding agents, which can significantly impact the results when the protein is used for aggregation studies and lead to false positive results as well. Therefore, this study provides insight into interaction of dye with crowding agent, which suggests taking appropriate blanks for protein aggregation studies.