Formation of amyloid fibrils underlies a wide range of human disorders, including Alzheimer's and prion diseases. The amyloid fibrils can be readily detected thanks to thioflavin T (ThT), a small molecule that gives strong fluorescence upon binding to amyloids. Using the amyloid fibrils of Aβ40 and Aβ42 involved in Alzheimer's disease, and of yeast prion protein Ure2, here we study three aspects of ThT binding to amyloids: quantification of amyloid fibrils using ThT, the optimal ThT concentration for monitoring amyloid formation and the effect of ThT on aggregation kinetics. We show that ThT fluorescence correlates linearly with amyloid concentration over ThT concentrations ranging from 0.2 to 500 µM. At a given amyloid concentration, the plot of ThT fluorescence versus ThT concentration exhibits a bell-shaped curve. The maximal fluorescence signal depends mostly on the total ThT concentration, rather than amyloid to ThT ratio. For the three proteins investigated, the maximal fluorescence is observed at ThT concentrations of 20–50 µM. Aggregation kinetics experiments in the presence of different ThT concentrations show that ThT has little effect on aggregation at concentrations of 20 µM or lower. ThT at concentrations of 50 µM or more could affect the shape of the aggregation curves, but this effect is protein-dependent and not universal.

In yeast, the formation of Ure2 fibrils underlies the prion state [URE3], in which the yeast loses the ability to distinguish good nitrogen sources from bad ones. The Ure2 prion domain is both necessary and sufficient for the formation of amyloid fibrils. Understanding the structure of Ure2 fibrils is important for understanding the propagation not only of the [URE3] prion but also of other yeast prions whose prion domains share similar features, such as the enrichment of asparagine and glutamine residues. Here, we report a structural study of the amyloid fibrils formed by the Ure2 prion domain using sitedirected spin labeling and electron paramagnetic resonance (EPR) spectroscopy. We completed a spin label scanning of all the residue positions between 2 and 80 of the Ure2 prion domain. The EPR data show that the Ure2 fibril core consists of residues 8−68 and adopts a parallel in-register β-sheet structure. Most of the residues show strong spin−exchange interactions, suggesting that there are only short turns and no long loops in the fibril core. Based on the strength of spin−exchange interactions, we determined the likely locations of the β-strands. EPR data also show that the C-terminal region of the Ure2 prion domain is more disordered than the N-terminal region. The roles of hydrophobic and charged residues are analyzed. Overall, the structure of Ure2 fibrils appears to involve a balance of stabilizing interactions, such as asparagine ladders, and destabilizing interactions, such as stacking of charged residues.