Heterogeneous amyloid β dimerization revealed by single-molecule FRET and MD simulation.

The correlation between specific amyloid fibril polymorphs and diseases suggests that characterization of heterogeneity in the aggregation process is key to understanding disease mechanisms. In this work, we use single-molecule Förster resonance energy transfer (FRET) spectroscopy and molecular dynamics (MD) simulation to quantitatively characterize dimerization of amyloid β (Aβ), the first step of the aggregation process. We find that dimerization is extremely rare, with a high dissociation constant of >16 mM, yet there are multiple dimer states with distinct FRET efficiencies and stabilities, many of which survive for milliseconds to seconds. The dimer population increases with the increasing ionic strength and decreasing pH, indicating the importance of nonspecific electrostatic interactions in dimer formation. MD simulations with enhanced sampling demonstrate the formation of diverse bound states having a range of FRET efficiencies and flexible N termini, compatible with the experimental observations. There are conformational clusters represented by the formation of three mixed parallel/antiparallel β strands forming Greek key-like motifs and the formation of antiparallel β strands, which may be representative of dimers with longer and shorter lifetimes, respectively, observed in the FRET experiment. This structural and kinetic heterogeneity, resulting from transient interactions, reflects the rugged energy landscape of amyloid formation and may be related to the great diversity of fibrils ultimately formed in the aggregation process.