Oligomers and fibrils of the amyloid-β (Aβ) peptide are implicated in the pathology of Alzheimer’s disease. Here, we monitor the growth of individual Aβ40 fibrils by time-resolved in situ atomic force microscopy and thereby directly measure fibril growth rates. The measured growth rates in a population of fibrils that includes both single protofilaments and bundles of filaments are independent of the fibril thickness, indicating that cooperation between adjacent protofilaments does not affect incorporation of monomers. The opposite ends of individual fibrils grow at similar rates. In contrast to the “stop-and-go” kinetics that has previously been observed for amyloid-forming peptides, growth and dissolution of the Aβ40 fibrils are relatively steady for peptide concentration of 0–10 μM. The fibrils readily dissolve in quiescent peptide-free solutions at a rate that is consistent with the microscopic reversibility of growth and dissolution. Importantly, the bimolecular rate coefficient for the association of a monomer to the fibril end is significantly smaller than the diffusion limit, implying that the transition state for incorporation of a monomer into a fibril is associated with a relatively high free energy.

Amyloid fibrillization is an exceedingly complex process in which incoming peptide chains bind to the fibril while concertedly folding. The coupling between folding and binding is not fully understood. We explore the molecular pathways of association of Aβ40 monomers to fibril tips by combining time-resolved in situ scanning probe microscopy with molecular modeling. The comparison between experimental and simulation results shows that a complex supported by nonnative contacts is present in the equilibrium structure of the fibril tip and impedes fibril growth in a supersaturated solution. The unraveling of this frustrated state determines the rate of fibril growth. The kinetics of growth of freshly cut fibrils, in which the bulk fibril structure persists at the tip, complemented by molecular simulations, indicate that this frustrated complex comprises three or four monomers in nonnative conformations and likely is contained on the top of a single stack of peptide chains in the fibril structure. This pathway of fibril growth strongly deviates from the common view that the conformational transformation of each captured peptide chain is templated by the previously arrived peptide. The insights into the ensemble structure of the frustrated complex may guide the search for suppressors of Aβ fibrillization. The uncovered dynamics of coupled structuring and assembly during fibril growth are more complex than during the folding of most globular proteins, as they involve the collective motions of several peptide chains that are not guided by a funneled energy landscape.

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