Two-step FRET as a tool for probing the amyloid state of proteins

Amyloid fibrils, a special class of protein aggregates with a core beta-sheet structure, are currently associated with a number of human disorders. The fluorescence-based techniques play essential role in detection and structural characterization of amyloid assemblies. The amyloid-sensing potential of the two-step Forster resonance energy transfer has been assessed using the three-chromophore system containing a benzothiazole dye Thioflavin T, a phosphonium dye TDV and a squaraine dye SQ1. The WT and amyloidogenic variants of the N-terminal fragment of apolipoprotein A-I with varying degrees of fibrillization have served as a scaffold for the cascade energy transfer ThT -> TDV -> SQ1. The FRET efficiencies in the donor-acceptor pairs ThT-TDV and TDV-SQ1 have been found to correlate with the extent of amyloid formation. It has been demonstrated that the two-step FRET format has the advantages relative to the conventional one-step FRET, among which are the large Stokes shift enabling the acquisition of the well-resolved 3D fluorescence patterns and a higher amyloid detection sensitivity. (C) 2019 Elsevier B.V. All rights reserved.