Unprecedented binding of Thioflavin T with well-ordered spherical aggregates: A false positive?

Amyloidogenic protein aggregation is a hallmark of numerous neurodegenerative diseases, including Alzheimer's and Parkinson's diseases. Thioflavin T (ThT), which selectively interacts with fibrillar amyloid structures, holds significant promise for diagnostic and therapeutic applications. Herein, we investigate the binding behaviour of Thioflavin T (ThT), a widely employed amyloid-specific fluorophore, with well-ordered spherical aggregates formed by dipeptides Boc-Phe-Trp-OMe (FW), Boc-Val-Trp-OMe (VW), Boc-Leu-Trp-OMe (LW) and Boc-Ile-Trp-OMe (IW). Our findings indicate that despite their non-amyloid nature, the well-ordered spherical dipeptide aggregates effectively sequester ThT molecules, enhancing fluorescence. The binding of ThT molecules to the dipeptides was further confirmed by fluorescence microscopy, which produced beautiful bright green-fluorescent images from the spherical structures and also with DFT studies. The apparent binding constant calculation suggests a reasonably good binding affinity between ThT and the designed dipeptide molecules, and the thermodynamic parameters analysis indicates the spontaneity of the binding process during complexation. The spherical nature of the dipeptides was confirmed by FESEM and FETEM. Circular Dichroism (CD) and Solid-state FTIR studies suggest that the dipeptides in solution coexist in multiple conformations. This study underscores the universality of ThT as a probe for fibrillar aggregate, sheds light on the broader implications of molecular recognition, and highlights the importance of investigating unexpected interactions in supramolecular chemistry and peptide-based materials. This is the first report of a ThT-stained spherical supramolecular structure made up of standard amino acids.