Structural properties of Aβ protofibrils stabilized by a small molecule

Metastable oligomeric and protofibrillar forms of amyloidogenic proteins have been implicated as on-pathway assembly intermediates in amyloid formation and as the major toxic species in a number of amyloid diseases including Alzheimer's disease. We describe here a chemical biology approach to structural analysis of A beta protofibrils. Library screening yielded several molecules that stimulate A beta aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts A beta(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37 degrees C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-A beta aggregates exhibit a low thioflavin T response. Like A beta fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-A beta aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous A beta fibril formation reaction: approximate to 12 of the 39 A beta(1-40) backbone amide protons are protected from exchange in the protofibril, compared with approximately twice that number in amyloid fibrils. Scanning proline mutagenesis analysis shows that the A beta molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in A beta conformation between fibrils and protofibrils is added structural definition in the 22-29 segment in the fibril. Besides aiding structural analysis, compounds capable of facilitating oligomer and protofibril formation might have therapeutic potential, if they act to sequester A beta in a form and/or location that cannot engage the toxic pathway.