Abstract
Transthyretin (TTR) amyloidosis is a progressive disease characterized by an abrupt aggregation of misfolded protein in multiple organs and tissues TTR is a tetrameric protein expressed in the liver and choroid plexus. Protein misfolding triggers monomerization of TTR tetramers. Next, monomers assemble forming oligomers and fibrils. Although the secondary structure of TTR fibrils is well understood, there is very little if anything is known about the structural organization of TTR oligomers. To end this, we used nano-infrared spectroscopy, also known as atomic force microscopy infrared (AFM-IR) spectroscopy. This emerging technique can be used to determine the secondary structure of individual amyloid oligomers and fibrils. Using AFM-IR, we examined the secondary structure of TTR oligomers formed at the early (3-6 h), middle (9-12 h), and late (28 h) of protein aggregation. We found that aggregating, TTR formed oligomers (Type 1) that were dominated by α-helix (40%) and β-sheet (~30%) together with unordered protein (30%). Our results showed that fibril formation was triggered by another type of TTR oligomers (Type 2) that appeared at 9 h. These new oligomers were primarily composed of parallel β-sheet (55%), with a small amount of antiparallel β-sheet, α-helix, and unordered protein. We also found that Type 1 oligomers were not toxic to cells, whereas TTR fibrils formed at the late stages of protein aggregation were highly cytotoxic. These results show the complexity of protein aggregation and highlight the drastic difference in the protein oligomers that can be formed during such processes.