Characterization of heterogeneous dimer and oligomer formation of amyloid-beta using single-molecule FRET.

Abstract

Protein aggregation is implicated as the cause of pathology in various diseases. Polymorphism in the structure of fibrils formed by aggregation suggests the existence of many different assembly pathways and therefore a heterogeneous ensemble of soluble oligomers. Characterization of this heterogeneity is the key to understanding the aggregation mechanism and toxicity of specific oligomers, but in practice it is extremely difficult to probe individual aggregation pathways in a mixture. Using fluorescence lifetime imaging and deep learning, we have recently visualized the formation of highly diverse fibrils of amyloid-beta (Abeta) in real time (PNAS_2022_e2116736119). Here, we further characterize the heterogeneity in the early stages of aggregation, dimerization and oligomerization of 42-residue Abeta (Abeta42), using single-molecule FRET. We found the dimerization of Abeta is extremely rare. The dimer dissociation constant is unexpectedly high, >5 mM. In addition, the dimerization is highly heterogeneous in terms of the FRET efficiency and kinetics. At least 6 clusters of dimers were needed to fit the entire equilibrium dimerization trajectories. This structural and kinetic diversity suggests that the heterogeneous fibril formation starts from the beginning of the assembly, the dimerization step. Then, we sought the formation of larger stable oligomers of Abeta42 that appear during aggregation. Combination of peptide incubation in a plate reader, single-molecule free-diffusion FRET experiment, and a newly-developed maximum likelihood method based on the rigorous photon statistics and diffusion theory allows for the accurate characterization of stable oligomers appearing at various stages of aggregation. We found that the size of these oligomers ranges from 30- to 100-mer and their overall population is unexpectedly low, less than 1 nM, before and during aggregation of 1-µM Abeta42 peptide. The amounts of small oligomers such as dimers and tetramers are negligible.