Single-Molecule Fluorescence Spectroscopy of Heterogeneous
Aggregation of Amyloid-β
Hoi Sung Chung
Laboratory of Chemical Physics,
NIDDK, National institutes of Health
Bethesda, MD
Protein aggregation is implicated as the cause of pathology in various diseases such as Alzheimer’s and Parkinson’s disease. 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. We have investigated oligomerization and fibril formation of the 42-residue amyloid-β peptide (Aβ42) that is associated with Alzheimer’s disease. We have combined single-molecule FRET, fluorescence lifetime imaging and deep learning (FNet) for monitoring individual fibril formation in real time. We found that the concentration of oligomers, including dimers, is extremely low and that even the dimerization is highly heterogeneous structurally and kinetically. We characterized the heterogeneous fibril formation in terms of the number of cross-β subunits, elongation speed, growth polarity and conformation of fibrils. Tracking individual fibril formation and growth also leads to the discovery of a new general nucleation mechanism (termed heterogeneous secondary nucleation), where a fibril is formed on the surface of an oligomer with a different structure. Further experimental characterization of heterogeneity involving Aβ42 will be important for better understanding the disease mechanism and for the discovering targets for drug therapy.
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