Free and bound Thioflavin T molecules with ultrafast relaxation: implications for assessment of protein binding and aggregation

Fluorescent dye Thioflavin T (ThT) is a widely used probe for the detection of amyloid fibrils, which are protein aggregates involved in the pathogenesis of neurodegenerative disorders. Upon the formation of a complex with amyloids, the fluorescence quantum yield of ThT increases 1000-fold due to a dramatic reduction of the nonradiative decay rate. This is accompanied by a remarkable change of ThT fluorescence lifetime τ from ∼1 to ∼1000 ps, thus making it possible to assess ThT binding to different systems using τ as an indicator. However, when measuring ThT interaction with proteins, one can observe that the binding affinity determined from the ThT fluorescence intensity's dependence on protein concentration may be orders of magnitude lower than that determined using τ. Here we show that this discrepancy at least partly originates from a limited temporal resolution when determining the fluorescence lifetime of ThT in the ThT-protein system using the time-correlated single photon counting technique (TCSPC), which is usually characterized by a ∼100 ps instrument response function. This results in the situation when a small fraction (∼1%) of ThT molecules with a relatively slow decay (τ ∼ 1000 ps) completely disguises the impact of ThT molecules with an ultrafast decay (τ ∼ 1 ps) to the overall measured fluorescence decay curve. Moreover, using the femtosecond-resolved fluorescence up-conversion technique, we demonstrate that not only free ThT molecules but also a subpopulation of protein-bound ThT molecules exhibits fluorescence decay on a 1 ps timescale. The obtained results are of critical importance for a reliable interpretation of protein binding and aggregation experiments when using a ThT assay with a fluorescence lifetime determined by the TCSPC as an indicator. (letter)