Embedding the Amyloid β‐Peptide Sequence in Green Fluorescent Protein Inhibits Aβ Oligomerization

Protein fibril formation is related to fatal diseases. For example, Alzheimer’s disease (AD) is a progressive neurodegenerative disease that is characterized by the fibrillization of amyloid bpeptide (Ab), which accumulates in senile plaques. Ab peptides can self-assemble into soluble oligomers, protofibrils, and amyloid fibrils. All of these aggregated forms contain significant b-sheet structure; the mature fibrils have a particularly well-organized supramolecular b structure. Ab possesses two hydrophobic regions in the center (residues 17–21) and at the C terminus (residues 30–42) of its sequence, which might promote fibril formation. Various peptides that mimic the central sequence of Ab have been designed as fibrillization inhibitors. Indeed, these short peptides can bind to Ab and inhibit its fibrillogenesis, and a five-residue peptide containing proline has been shown to partially decrease amyloid deposition. Although amyloid fibrils were initially hypothesized to be the toxic species that drives AD, recent studies have imACHTUNGTRENNUNGplicated the soluble oligomeric species of Ab, which has been shown to kill neurons in cultured hippocampal brain slices. 9] However, it is not known whether the short peptides mentioned above can inhibit the generation of the toxic, soluble Ab oligomers. Recently, it was reported that antibodies against the Ab peptide can reduce Ab oligomers and their toxicity, which lends support to the therapeutic strategy of targeting Ab with molecules that bind it tightly. In contrast to Ab, green fluorescent protein (GFP) isolated from the jellyfish Aequorea victoria, is a soluble globular protein that generates a fluorophore autocatalytically. Hecht and co-workers constructed a fusion of Ab to GFP for screening compounds that inhibit Ab aggregation. GFP folds into a b-barrel structure composed of eleven b strands. The fluorophore is on an a helix that is buried in the center of the barrel. Both parallel and antiparallel b sheets are included in the GFP structure. By replacing some of the b strands in GFP with those from Ab, the protein not only contains the Ab sequence but also presents the b-sheet structure of Ab. Therefore, the protein construct should inhibit the oligomerization of Ab by binding to it. Further, since GFP has a very stable structure, the protein construct should not unfold or aggregate nonspecifically. In the present study, we designed and synthesized GFP variants in which two b strands derived from the Ab sequence were embedded in the sequence of GFP as an Ab structural mimic. These GFP variants can bind to Ab and inhibit its oligomerization. Models of both parallel and antiparallel b-sheet structures were utilized to construct the GFP variants. The model structures of Ab1–40 and Ab1–42 fibrils are composed of a parallel b-sheet organization. In contrast, the fibrils formed by residues 11–25 of Ab (Ab11–25) are composed of an antiparallel bsheet structure in the model. GFP contains eleven b strands, ten of which are aligned antiparallel and one that is oriented in a parallel fashion. The first and sixth strands of GFP are the parallel pair and thus were chosen for making a mimic of the parallel b-sheet model of Ab structure. A GFP variant named P13H was constructed, which contained the following mutations: P13H, L15K, E17V, D19F, D21E, T118H, V120K, R122V, E124F, and K126E. The first five mutations are located on strand 1 and the others on strand 6. The mutations were de-