Structural dynamics of thioflavin T during the intramolecular charge transfer probed by time-resolved Raman spectroscopy

Thioflavin T (ThT) is a well-known fluorophore used to visualize amyloid fibrils associated with Alzheimer’s disease. In polar solvents, efficient intramolecular charge transfer (ICT) quenches ThT’s fluorescence, while binding to amyloid fibrils inhibits the ICT, resulting in strong fluorescence. Structural changes of ThT, including the bend of benzothiazole and the twist of dimethylaniline, have been suggested in recent experimental and theoretical explorations of ICT. However, detailed experimental evidence for such structural changes remains limited. In this study, we report on the ICT dynamics of ThT using impulsive stimulated Raman spectroscopy (ISRS) and femtosecond stimulated Raman spectroscopy (FSRS). The population dynamics of the skeleton modes (1500-1600 cm^-1) of ThT from FSRS retrieved the coherent oscillations of ~100 and 196 cm^-1, suggesting anharmonic coupling of the ICT coordinate with low-frequency in-plane and out-of-plane deformation modes. Furthermore, ISRS results revealed two ICT dynamics (~370 fs and 3.4 ps) in the low-frequency vibrational modes (200-650 cm^-1) of ThT, suggesting that the structural changes of ThT occur in two steps: the bend of benzothiazole (~370 fs) followed by the twist of dimethylaniline (3.4 ps). Our experimental results, supported by the time-dependent density functional theory calculations, provide clear evidence for the multifaceted structural changes of ThT with ICT.