An Aggregate Weight-Normalized Thioflavin-T Measurement Scale for Characterizing Polymorphic Amyloids and Assembly Intermediates

Abstract

The red shift in the fluorescence excitation spectra of thioflavin dyes upon binding to fibrils has been a boon to the amyloid field, offering simple and effective methods for the qualitative detection of amyloid in tissue samples and for quantitation of particular fibril preparations with gravimetric linearity. The quantitative aspect of the thioflavin T (ThT) response, however, comes with an important caveat that bestows both significant limitations and great untapped power. It is now well established that amyloid fibrils of different proteins, as well as polymorphic fibrils of the same protein, can exhibit vastly different ThT fluorescence intensities for the same weight concentration of aggregates. Furthermore, the aggregated intermediates commonly observed in amyloid assembly reactions can exhibit aggregate weight-normalized (AWN) ThT fluorescence intensities that vary from essentially zero through a wide range of intermediate values before reaching the intensity of homogeneous, mature amyloid. These features make it very difficult to quantitatively interpret, without additional data, the time-dependent development of ThT fluorescence intensity in an assembly reaction. In this chapter, we describe a method for coupling ex situ ThT fluorescence determinations with an analytical HPLC supported sedimentation assay (also described in detail) that can provide significant new insights into amyloid assembly reactions. The time dependent aggregation data provided by the sedimentation assay reveals a time course of aggregation that is largely independent of aggregate properties. In addition, the combination of these data with ThT measurements of the same reaction time points reveals important aspects of average aggregate structure at each time point. Examples of the use and potential value of AWN-ThT measurements during amyloid assembly Aβ and polyglutamine peptides are provided.

Early Stage Alpha-Synuclein Amyloid Fibrils are Reservoirs of Membrane-Binding Species

“…It is well established, that the extensively used ThT-assay for detection of the formation of amyloid cannot be used quantitatively without caution 44 . Fibril polymorphs may bind different amounts of ThT and/or cause different fluorescence intensity, and intermediate, non-fibril aggregates may also contribute to the recorded fluorescence intensity.…”

“…Our report has several additional consequences, which are relevant to consider. It is well-known that the plateau in ThT fluorescent assays by no means can not automatically be considered as a stable phase of fibrillation 44 . Our results elaborate significantly on this notion, showing that under the disputably most commonly applied experimental conditions for αSN fibrillation, the solution is depleted from soluble material accompanied by a structural transformation between different fibril polymorphs, notably while both the ThT-signal and macroscopic appearance of the fibrils remain stable.…”

N-terminal mutants of human apolipoprotein A-I: structural perturbations associated to protein misfolding

“…Thioflavin T (ThT) is widely used to detect amyloid-like structures [24]. Although ThT-associated quantum yield usually increases proportionally to the formation of fibrillar protein aggregates, significant fluorescence is detected even when proteins are present as oligomeric conformations [25][26]. To address the effect of pH on the yield of amyloid-like protein aggregates, Wt (0.2 mg/mL) was taken for 48 h at 37°C at different pH between 7.4 and 5.0 in Citrate phosphate buffer, and ThT added at a molar ratio of two with respect to protein.…”

Peer Review #1 of "Exploring the potential of deep-blue autofluorescence for monitoring amyloid fibril formation and dissociation (v0.1)"

“…First, the denaturant can influence ThT affinity to fibrils and its fluorescence properties (Kuznetsova et al 2012;Voropai et al 2003). Moreover, the dye may be unable to cover the surface of amyloids that associate into larger aggregates during fibrilization (Wetzel et al 2018), and its ability to bind to oligomers or small protofibrils may be limited (Biancalana and Koide 2010). Furthermore, there are reports showing that ThT can modulate aggregation rates (Di Carlo et al 2015;Xue et al 2017).…”

“…Furthermore, there are reports showing that ThT can modulate aggregation rates (Di Carlo et al 2015;Xue et al 2017). Finally, it is known that long exposures to light (Wetzel et al 2018), elevated temperatures, and higher pH values can cause chemical modifications to ThT molecules, thus diminishing their use in lengthy real-time observation experiments.…”