Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40-and 42-Residue Amyloid-β

The two most abundant isoforms of amyloid-beta (A beta) are the 40- (A beta 40) and 42-residue (A beta 42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the A beta aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling A beta 42 and A beta 40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of A beta 42 with a small amount of A beta 40, while the second phase results mostly from aggregation of A beta 40. We also found that the aggregation of A beta 42 is slowed by A beta 40 while the aggregation of A beta 40 is accelerated by A beta 42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the A beta 42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of A beta 40 in the co-oligomers in a 1:1 mixture of A beta 42 and A beta 40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of A beta 42 and A beta 40 (1:10), suggesting the critical role of A beta 40 in oligomer formation and aggregation.