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

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Monoclonal antibodies were selected after immunization with crystals of the tripeptide l-leucine-l-leucine-l-tyrosine. They interact with the tripeptide crystals, but do not interact with the tripeptide molecule, with other crystalline surfaces, or with adsorbed protein. The interactions of two antibodies with crystals of l-Leu-l-Leu-l-Tyr and of its enantiomer d-Leu-d-Leu-d-Tyr were characterized in depth. Antibody 48E is stereoselective and enantioselective: it recognizes only the [011] faces of the l-Leu-l-Leu-l-Tyr crystals, and not the enantiomorphous [011] faces of d-Leu-d-Leu-d-Tyr crystals, or any other faces of either crystal. In contrast, antibody 602E is poorly stereoselective and is not enantioselective: it recognizes the crystals of both enantiomers, interacting with a number of different faces of each. The different recognition patterns are explained on the basis of the nature of the interactions and the structure of the interacting surfaces. Understanding this antibody specificity advances our general understanding of surface recognition and transfer of chiral information across biological interfaces.