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Thioflavin-T (ThT) is the most commonly used fluorescent dye for following amyloid formation semiquantitatively in vitro, specifically probing the fibrillar cross-b-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 a-synuclein polymorphs. In ThT fluorescence assays the two polymorphs show type-specific fluorescence intensity behaviour although their b-sheet content has been shown to be similar. Further, fluorescence lifetime measurements of fibrilbound 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.

Amyloid polymorphs have become one of the focal points of molecular studies of neurodegenerative diseases like Parkinson's disease. Due to their distinct biochemical properties and prion-like characteristics, insights into the molecular origin and stability of amyloid polymorphs over time are crucial for understanding the potential role of amyloid polymorphism in these diseases. Here, we systematically study the fibrillization of recombinantly produced human α-synuclein (αSyn) over an extended period of time to unravel the origin and temporal evolution of polymorphism. We follow morphological changes in the same fibril sample with atomic force microscopy over a period of 1 year. We show that wild-type (wt) αSyn fibrils undergo a slow maturation over time after reaching the plateau phase of aggregation (as detected in a Thioflavin-T fluorescence assay). This maturation, visualized by changes in the fibril periodicity over time, is absent in the disease mutant fibrils. The β-sheet content of the plateau phase and matured fibrils, obtained using Fourier transform infrared spectroscopy, is however similar for the αSyn protein sequences, suggesting that the morphological changes in wt αSyn fibrils are tertiary or quaternary in origin. Furthermore, results from a reversibility assay show that the plateau phase fibrils do not disassemble over time. Together, the observed changes in the periodicity distributions and stability of the fibrillar core over time point toward two distinct mechanisms that determine the morphology of wt αSyn fibrils: competitive growth between different polymorphs during the fibrillization phase followed by a process wherein fibrils undergo slow maturation or annealing.

Under aggregation-prone conditions, soluble amyloidogenic protein monomers can self-assemble into fibrils or they can fibrillize on preformed fibrillar seeds (seeded aggregation). Seeded aggregations are known to propagate the morphology of the seeds in the event of cross-seeding. However, not all proteins are known to cross-seed aggregation. Cross-seeding has been proposed to be restricted either because of differences in the protein sequences or because of conformations between the seeds and the soluble monomers. Here, we examine cross-seeding efficiency between three α-synuclein sequences, wild-type, A30P, and A53T, each varying in only one or two amino acids but forming morphologically distinct fibrils. Results from bulk Thioflavin-T measurements, monomer incorporation quantification, single fibril fluorescence microscopy, and atomic force microscopy show that under the given solution conditions conformity between the conformation of seeds and monomers is essential for seed elongation. Moreover, elongation characteristics of the seeds are defined by the type of seed.