No abstract

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␤ protofibrils. Library screening yielded several molecules that stimulate A␤ aggregation. One of these compounds, calmidazolium chloride (CLC), rapidly and efficiently converts A␤(1-40) monomers into clusters of protofibrils. As monitored by electron microscopy, these protofibrils persist for days when incubated in PBS at 37°C, with a slow transition to fibrillar structures apparent only after several weeks. Like normal protofibrils, the CLC-A␤ aggregates exhibit a low thioflavin T response. Like A␤ fibrils, the clustered protofibrils bind the anti-amyloid Ab WO1. The CLC-A␤ aggregates exhibit the same protection from hydrogen-deuterium exchange as do protofibrils isolated from a spontaneous A␤ fibril formation reaction: Ϸ12 of the 39 A␤(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␤ molecule in these protofibrillar assemblies exhibits the same flexible N and C termini as do mature amyloid fibrils. The major difference in A␤ 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␤ in a form and͞or location that cannot engage the toxic pathway.amyloid ͉ chemical biology ͉ hydrogen exchange ͉ proline scanning

Gradual corrosion of stainless steel electrospray emitters under conditions of normal use generates surface irregularities that can promote electrical discharge. The increased emission current affects the electrochemical reactions associated with the spray process. When sampling the peptide Aβ(1-40), this is manifest by oxidation of methionine at position 35 to methionine sulfoxide. The resultant mass shift and reduced sensitivity can adversely affect H/D exchange experiments. These effects can be avoided by adding a redox buffer or (preferably) by re-polishing the emitter, especially to a rounded geometry.