Pentameric Thiophene as a Probe to Monitor EGCG's Remodeling Activity of Mature Amyloid Fibrils: Overcoming Signal Artifacts of Thioflavin T

Abstract

Thioflavin T fluorescence is a gold standard probe for the detection of amyloid fibrils. Herein, we showed that mature amyloid fibrils incubated with polyphenol epigallocatechin gallate (EGCG) present a fast reduction of the thioflavin T fluorescence, which is not related to remodeling activity. We propose the use of the pentameric thiophene fluorescence for monitoring the polyphenol remodeling activity.

Figure 1

Effect of EGCG on the ThT and PFTAA fluorescence of α-synuclein amyloid fibrils. (A) Experimental scheme of the centrifugation/wash protocol. (B) ThT fluorescence or (C) PFTAA fluorescence of the samples incubated for 0 or 24 h in the absence or presence of EGCG. The use of the centrifugation/wash protocol is denoted by (+). The buffer used for all assays was 50 mM phosphate buffer pH 7.4, 150 mM NaCl (25 °C). [α-synuclein] = 5 μM and [EGCG] = 30 μM. For the ThT fluorescence, the ThT concentration used was 20 μM, Ex: 450 nm, and Em: 477 nm. For the PFTAA fluorescence, the PFTAA concentration used was 20 μM, Ex: 450 nm, and Em: 550 nm. Transmission electron microscopy of α-synuclein amyloid fibrils incubated in the absence (D) or presence of EGCG for 0 (E) or 24 h (F). Bar = 500 nm.

Figure 2

Titration of EGCG at α-synuclein amyloid fibrils monitored by ThT or PFTAA. Mature α-synuclein amyloid fibrils (10 μM) were incubated with 20 μM ThT (A) or PFTAA (B), and the fluorescence was measured (green spectra). A titration of EGCG ranging from 5 to 25 μM was performed, and the fluorescence of ThT or PFTAA was measured immediately. As a negative control, no fibrils were added to the ThT or PFTAA solution (black spectra). Panel C presents the area of each spectrum at increasing EGCG concentrations.

Figure 3

Titration of amyloidogenic probes at α-synuclein amyloid fibrils monitored incubated with EGCG. Mature α-synuclein amyloid fibrils (10 μM) incubated with different concentrations of ThT (A) or PFTAA (B) were incubated with increasing concentrations of EGCG, and their fluorescence was measured at zero time point.

Figure 4

Kinetics of α-synuclein amyloid fibril remodeling monitored by ThT or PFTAA fluorescence incubated with EGCG. α-synuclein amyloid was incubated in the absence (open symbols) or presence of EGCG (hatched symbols) at 37 °C, and the ThT (circles) or PFTAA (squares) fluorescence was monitored according to the time. [α-synuclein] = 5 μM and [EGCG] = 30 μM. For the ThT fluorescence, the ThT concentration used was 20 μM, Ex: 450 nm, and Em: 477 nm. For the PFTAA fluorescence, the PFTAA concentration used was 20 μM, Ex: 450 nm, and Em: 550 nm.