Variable Binding of Thioflavin T by Amyloid Fibrils

Abstract

Thioflavin T (ThioT) fluorescence is commonly used to quantify amyloid formation in vivo and in vitro, but its binding mode and basis for fibril selectivity is not understood, and there is no consensus on the relationship between fluorescence intensity and fibril mass. To determine whether the rate of fibril formation affected ThioT fluorescence, fibrils of amyloid beta (Aβ) proteins were formed at different concentrations of the monomeric protein. The intensity of ThioT fluorescence increased with the monomer concentration during fibril formation. A similar dependence on monomer concentration was observed for insulin fibrils. The affinity of the dye for Aβ fibrils formed at different rates was assessed by surface-plasmon resonance (SPR), indicating that fibrils grown from more concentrated monomer solutions bound larger amounts of ThioT per mass of Aβ, consistent with the steady-state fluorescence results. However, the dissociation constants and the affinity of individual binding sites were independent of the monomer concentration of Aβ proteins. The orientation of ThioT in Aβ fibrils was evaluated with polarized fluorescence microscopy, and found to be aligned toward the long axis of the fibril. On the basis of these results, it is proposed that ThioT binds to localized polymorphic sites along the length of a fibril, rather than to well-defined binding sites in a mature fibril with regular structure.