Interaction of Thioflavin T (ThT) and 8-anilino-1-naphthalene sulfonic acid (ANS) with macromolecular crowding agents and their monomers: Biophysical analysis using in vitro and computational approaches

Protein aggregation is a major side reaction that may be observed during protein folding and may prevent proteins from performing their intended functions. It is a hallmark of various human diseases, specifically neurodegenerative diseases. Fluorescence spectroscopy is one such technique in which extrinsic fluorescent dyes are used to monitor protein aggregation in vitro. These dyes are known to get bound to the aggregated portion of proteins, which thereby leads to an enhancement of fluorescence intensity. In this study, we have uniquely investigated with a battery of evidence to show that extrinsic fluorescent dyes bind to macromolecular crowding agents and their monomers even in absence of protein. The additives used in this study are dextran-70 and ficoll-70 and their monomers, glucose and sucrose, respectively. Here, with the help of various biophysical techniques such as extrinsic fluorescent dye-binding assays, molecular docking, ITC and TEM, we have explored the interactions that exist between these extrinsic fluorescent dyes and the macromolecular crowding agents along with their monomers. This study inferred that there is a high possibility of interaction between dyes and additives, as these dyes showed strong binding with above-mentioned crowding agents and their monomers.