According to the amyloid hypothesis, the pathogenesis of Alzheimer's disease is triggered by the oligomerization and aggregation of the amyloid-␤ (A␤) peptide into protein plaques. Formation of the potentially toxic oligomeric and fibrillar A␤ assemblies is accompanied by a conformational change toward a high content of ␤-structure. Here, we report the solution structure of A␤(1-40) in complex with the phage-display selected affibody protein ZA␤3, a binding protein of nanomolar affinity. Bound A␤(1-40) features a ␤-hairpin comprising residues 17-36, providing the first highresolution structure of A␤ in ␤ conformation. The positions of the secondary structure elements strongly resemble those observed for fibrillar A␤. ZA␤3 stabilizes the ␤-sheet by extending it intermolecularly and by burying both of the mostly nonpolar faces of the A␤ hairpin within a large hydrophobic tunnel-like cavity. Consequently, Z A␤3 acts as a stoichiometric inhibitor of A␤ fibrillation. The selected A␤ conformation allows us to suggest a structural mechanism for amyloid formation based on soluble oligomeric hairpin intermediates.A␤-peptide ͉ engineered binding protein ͉ molecular recognition ͉ protein structure ͉ nuclear magnetic resonance

We describe the development of a novel serum albumin binding protein showing an extremely high affinity (K(D)) for HSA in the femtomolar range. Using a naturally occurring 46-residue three-helix bundle albumin binding domain (ABD) of nanomolar affinity for HSA as template, 15 residues were targeted for a combinatorial protein engineering strategy to identify variants showing improved HSA affinities. Sequencing of 55 unique phage display-selected clones showed a strong bias for wild-type residues at nine positions, whereas various changes were observed at other positions, including charge shifts. Additionally, a few non-designed substitutions appeared. On the basis of the sequences of 12 variants showing high overall binding affinities and slow dissociation rate kinetics, a set of seven 'second generation' variants were constructed. One variant denoted ABD035 displaying wild-type-like secondary structure content and excellent thermal denaturation/renaturation properties showed an apparent affinity for HSA in the range of 50-500 fM, corresponding to several orders of magnitude improvement compared with the wild-type domain. The ABD035 variant also showed an improved affinity toward serum albumin from a number of other species, and a capture experiment involving human serum indicated that the selectivity for serum albumin had not been compromised from the affinity engineering.