Steady-state and time-resolved Thioflavin-T fluorescence can report on morphological differences in amyloid fibrils formed by Aβ(1-40) and Aβ(1-42)

Abstract

Thioflavin-T (ThT) is one of the most commonly used dyes for amyloid detection, but the origin of its fluorescence enhancement is not fully understood. Herein we have characterised the ThT fluorescence response upon binding to the Aβ(1-40) and Aβ(1-42) variants of the Alzheimer's-related peptide amyloid-β, in order to explore how the photophysical properties of this dye relates to structural and morphological properties of two amyloid fibril types formed by peptides with a high degree of sequence homology. We show that the steady-state ThT fluorescence is 1.7 times more intense with Aβ(1-40) compared to Aβ(1-42) fibrils in concentration matched samples prepared under quiescent conditions. By measuring the excited state lifetime of bound ThT, we also demonstrate a distinct difference between the two fibril isoforms, with Aβ(1-42) fibrils producing a longer ThT fluorescence lifetime compared to Aβ(1-40). The substantial steady-state intensity difference is therefore not explained by differences in fluorescence quantum yield. Further, we find that the ThT fluorescence intensity, but not the fluorescence lifetime, is dependent on the fibril preparation method (quiescent versus agitated conditions). We therefore propose that the fluorescence lifetime is inherent to each isoform and sensitively reports on fibril microstructure in the protofilament whereas the total fluorescence intensity relates to the amount of exposed β-sheet in the mature Aβ fibrils and hence to differences in their morphology. Our results highlight the complexity of ThT fluorescence, and demonstrate its extended use in amyloid fibril characterisation.

Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid β Aggregation

“…ThT is a fluorescent dye which preferentially binds to β -sheet-rich amyloid aggregates, yielding an increase in fluorescence intensity. 42,43 A 3-fold molar excess (75 µ M) of polymer was incubated with A β (25 µ M) in 20 mM Tris at pH 8.0 with 0.1 M NaNO 3 and 0.01% NaN 3 . Samples were kept at 37 °C, and ThT fluorescence was measured periodically (Figure 3A–C).…”

Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding

“…One possibility is that the amyloid formation reactions with FL proteins do not have the same amount of amyloid formed at the end of the reactions. The other explanation is that the structure of the amyloid formed by FL proteins does not allow as many ThT binding sites as the structure for V L amyloid or that the affinity of each binding site is different [19]. In addition, it is possible that ThT molecules bound to the different fibrils have different fluorescence quantum yields.…”

C-Terminal Threonine Reduces Aβ43 Amyloidogenicity Compared with Aβ42

“…In situations where there is effectively only one aggregated species (such as the end of the reaction) the weight normalized ThT value is characteristic of the polymorphic state of the aggregate. Previously we showed that structurally different polymorphic forms of mature Aβ 40 fibrils exhibit different weight-normalized ThT intensities 39 , and it was recently suggested that such intensity variations are due to differences in the number of ThT binding sites (normally considered to be in β-sheet 40 ) in an aggregate 41 .…”