Unravelling the non-classical nucleation mechanism of an amyloid nanosheet through atomic force microscopy and an...

Understanding the amyloid nucleation mechanism is fundamentally important for the development of diagnostics and therapeutics of amyloid-related diseases and for the design and application of amyloid-based materials. To this end, we here explore the use of atomic force microscopy (AFM) and a side-chain-based infrared (IR) probe technique to investigate the amyloid nanosheet formation mechanism of an Aβ_16-22 variant, KLVFXAK, where X is p-cyanophenylalanine with its side-chain cyano group being an infrared probe. Using AFM, we reveal that the formation of KLVFXAK amyloid nanosheets follows a two-step non-classical nucleation mechanism. The first step is the rapid formation of a metastable fibrillar intermediate and the second step is slow transformation to the final nanosheet. Using the side-chain-based IR probe technique, we obtain spectroscopic evidence for the proposed nucleation mechanism of the amyloid nanosheet as well as the structural details for the intermediate and amyloid nanosheet. By using the structural constraints set by the two techniques, we propose the structural models for both the fibrillar intermediate and the amyloid nanosheet. In addition, we further investigated the amyloid nanosheet formation mechanism of a similar Aβ₁₆ variant, KLVFXAE, and showed the impact of mutation on the amyloid nucleation mechanism. Our work also provides a nice example of how to use the combined approach of AFM and a side-chain-based IR probe technique to unravel the complex nucleation mechanism of amyloid formation.