Evidence for a Mechanism of Amyloid Formation Involving Molecular Reorganisation within Native-like Precursor Aggregates

The aggregation of the α/β protein acylphosphatase from Sulfolobus solfataricus has been studied under conditions in which the protein maintains a native-like, although destabilised, conformation and that therefore bear resemblance to a physiological medium. Static and dynamic light-scattering measurements indicate that under these conditions the protein aggregates rapidly, within two minutes. The initial aggregates are enzymatically active and have a secondary structure that is not yet characterized by the high content of cross-β structure typical of amyloid, as inferred from Fourier transform infra-red and circular dichroism measurements. These species then convert slowly into enzymatically inactive aggregates that bind thioflavin T and Congo red, characteristic of amyloid structures, and contain extensive β-sheet structure. Transmission electron microscopy reveals the presence in the latter aggregates of spherical species and thin, elongated protofibrils, both with diameters of 3–5 nm. Kinetic tests reveal that this process occurs without the need for dissolution and re-nucleation of the aggregates. Formation of thioflavin T-binding and β-structured aggregates is substantially more rapid than unfolding of the native state, indicating that the initial aggregation process promotes formation of amyloid structures. Taken together, these findings suggest a mechanism of amyloid formation that may have physiological relevance and in which the amyloid structures result from reorganisation of the molecular interactions within the initially formed non-amyloid aggregates.