Heterogeneity and Turnover of Intermediates during Amyloid-β (Aβ) Peptide Aggregation Studied by Fluorescence Correlation Spectroscopy

Self-assembly of amyloid beta (A beta) peptide molecules into large aggregates is a naturally occurring process driven in aqueous solution by a dynamic interplay between hydrophobic interactions among A beta molecules, which promote aggregation, and steric and overall electrostatic hindrance, which stifles it. A beta self-association is entropically unfavorable, as it implies order increase in the system, but under favorable kinetic conditions, the process proceeds at appreciable rates, yielding A beta aggregates of different sizes and structures. Despite the great relevance and extensive research efforts, detailed kinetic mechanisms underlying A beta aggregation remain only partially understood. In this study, fluorescence correlation spectroscopy (FCS) and Thioflavin T (ThT) were used to monitor the time dependent growth of structured aggregates and characterize multiple components during the aggregation of A beta peptides in a heterogeneous aqueous solution. To this aim, we collected data during a relatively large number of observation periods, 30 consecutive measurements lasting 10 s each, at what we consider to be a constant time point in the slow aggregation process. This approach enabled monitoring the formation of nanomolar concentrations of structured amyloid aggregates and demonstrated the changing distribution of amyloid aggregate sizes throughout the aggregation process. We identified aggregates of different sizes with molecular weight from 260 to more than 1 x 10(6) kDa and revealed the hitherto unobserved kinetic turnover of intermediates during A beta aggregation. The effect of different A beta concentrations, A beta:ThT ratios, differences between the 40 (A beta 40) and 42 (A beta 42) residue long variants of A beta, and the effect of stirring were also examined.