The three-step Förster resonance energy transfer (FRET) within the cascade of four dyes, including the classical amyloid marker Thioflavin T as a primary donor, two jumper dyes, benzanthrone ABM and squaraine SQ4, and terminal acceptor SQ1, was tested as a possible tool for detection and characterization of insulin amyloid fibrils. The results obtained confirm the occurrence of highly efficient multistep FRET (msFRET) in the chromophore ensemble in the presence of insulin fibrils formed at elevated temperature under pH 2 (InsF1) or pH 7.4, 0.15 M NaCl (InsF2), while negligible FRET efficiencies were obtained for the control unfibrillized protein, suggesting the specificity of msFRET to cross-β-sheet architecture characteristic of amyloid fibrils. Specifically, the efficiencies of FRET for the donor-acceptor pairs ThT-ABM, ABM-SQ4 and SQ4-SQ1 at maximum acceptor concentrations (~0.4 µM – 1.6 µM) were estimated to be 86%/94%, 48%/34% and 66%/32%, respectively, in the presence of InsF1/InsF2. The most significant differences between InsF1/InsF2 and the control protein were observed for the donor-acceptor pair ThT-ABM, suggesting that ABM is the key mediator in the whole process of msFRET. Assuming the isotropic rotation of the fluorophores, the average donor-acceptor distances were estimated in the presence of InsF1, yielding the values 1.3 nm, 5.3 nm, and 3.9 nm for the ThT-ABM, ABM-SQ4 and SQ4-SQ1 pairs, respectively. The obtained distances are indicative of different fibril binding sites for the chromophores in the insulin fibrils, although due to their high specificity to the fibrillar structure, the dyes are most likely to localize in the surface grooves of β-sheets running along the main axis of amyloid fibril. Remarkably, the differences in the insulin amyloid morphology can be clearly distinguished using msFRET. As evidenced from TEM, InsF2 were thinner, shorter and contained amorphous aggregates, as compared to InsF1. Thus, different amyloid formation pathways under neutral and acidic pH resulted in the changes in the dye affinity for to the fibril binding sites, and, as a consequence, in the distinct msFRET efficiencies, especially for the pair SQ4-SQ1. The ability of ThT to serve as an efficient amplifier for the two near-infrared dyes, SQ4 and SQ1, with the benzanthrone fluorophore ABM as a jumper dye, allows detection of fibrillar insulin in the optical window of the biological samples, with the Stokes shift of the four-chromophore being ca. 240 nm. The proposed msFRET-based approach can be employed not only for insulin amyloid detection but also for distinguishing between different amyloid fibril morphologies and gaining further insights into the mechanisms involved in the development of the injection-localized insulin amyloidosis.
Ultra pseudo-Stokes shift near infrared dyes based on energy transfer