Amyloid formation under physiological conditions proceeds via a native-like folding intermediate

Although most proteins can assemble into amyloid-like fibrils in vitro under extreme conditions, how proteins form amyloid fibrils in vivo remains unresolved. Identifying rare aggregation-prone species under physiologically relevant conditions and defining their structural properties is therefore an important challenge. By solving the folding mechanism of the naturally amyloidogenic protein β-2-microglobulin at pH 7.0 and 37 °C and correlating the concentrations of different species with the rate of fibril elongation, we identify a specific folding intermediate, containing a non-native trans -proline isomer, as the direct precursor of fibril elongation. Structural analysis using NMR shows that this species is highly native-like but contains perturbation of the edge strands that normally protect β-sandwich proteins from self-association. The results demonstrate that aggregation pathways can involve self-assembly of highly native-like folding intermediates, and have implications for the prevention of this, and other, amyloid disorders.