Polymorph-specific distribution of binding sites determines thioflavin-T fluorescence intensity in α-synuclein fibrils

Abstract

Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semi-quantitatively in vitro, specifically probing the fibrillar cross-β-sheet content. In recent years, structural polymorphism of amyloid fibrils has been shown to be an important aspect of amyloid formation, both in vitro and in neurodegenerative diseases. Therefore, understanding ThT–amyloid interactions in the context of structural polymorphism of amyloids is necessary for correct interpretation of ThT fluorescence data. Here we study the influence of fibril morphology on ThT fluorescence and ThT binding sites, with two morphologically distinct but chemically identical α-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their β-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibril-bound ThT reveal the presence of at least two qualitatively different ThT binding sites on the polymorphs. The relative distributions of the binding sites on the fibril surfaces appear to be morphology dependent, thus determining the observed polymorph-specific ThT fluorescence intensities. These results, highlighting the role of fibril morphology in ThT-based amyloid studies, underline the relevance of polymorphs in ThT–amyloid interaction and can explain the variability often observed in ThT amyloid binding assays.

Keywords:

• Amyloid
• polymorphism
• α-synuclein
• thioflavin-T
• atomic force microscopy

Acknowledgements

The authors thank Kirsten van Leijenhorst-Groener, Yvonne Kraan and Nathalie Schilderink for protein expression and purification, and Dr Martin Bennink, Kees van der Werf and Robert Molenaar for advice on AFM.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is supported by NanoNextNL, a micro- and nanotechnology consortium of the Government of The Netherlands and 130 partners.