An efficient method to generate fluorescent amyloid fibrils

Studies on fluorophore-tagged peptides help in elucidating the molecular mechanism of amyloidogenesis including their cellular internalization and crosstalk potential. Despite several advantages, unavoidable difficulties including expensive and tedious synthesis-protocols exist in fluorophore-based tools. Importantly, covalently-tagged fluorophores could introduce structural constraints which may influence the conformation of the monomeric and aggregated forms of protein. To resolve this problem, we describe a robust yet simple method to make fluorescent amyloid fibrils through non-covalent incorporation of fluorophores into amyloid fibrils. We used aggregation protocol in which a small amount of fluorophore is incorporated into the amyloids, and this protocol does not alter the aggregation kinetics and the characteristic {beta}-sheet-conformers of the generated amyloid fibrils. We have successfully prepared fluorescent amyloid fibrils of Insulin, Lysozyme and A{beta}1-42, and the noncovalently incorporated fluorophores remained intact in the amyloid fibrils without leaching, even after serial-dilutions and prolonged-storage. Further, this method enables successful monitoring of cellular-internalization of the fluorescent amyloids into SH-SY5Y and A549 cells, and it also detects FRET-signals during interfibrillar interactions. The findings establish a simple and affordable protocol to prepare fluorescent amyloid structures, which may significantly help amyloid researchers working on both in vitro and animal model systems.