Rational design of amyloid beta peptide-binding proteins: pseudo-Abeta beta-sheet surface presented in green fluorescent protein binds tightly and preferentially to structured Abeta.
Some neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease are caused by protein misfolding. In AD, amyloid beta-peptide (Abeta) is thought to be a toxic agent by self-assembling into a variety of aggregates involving soluble oligomeric intermediates and amyloid fibrils. Here, we have designed several green fluorescent protein (GFP) variants that contain pseudo-Abeta beta-sheet surfaces and evaluated their abilities to bind to Abeta and inhibit Abeta oligomerization. Two GFP variants P13H and AP93Q bound tightly to Abeta, K(d) = 260 nM and K(d) = 420 nM, respectively. Moreover, P13H and AP93Q were capable of efficiently suppressing the generation of toxic Abeta oligomers as shown by a cell viability assay. By combining the P13H and AP93Q mutations, a super variant SFAB4 comprising four strands of Abeta-derived sequences was designed and bound more tightly to Abeta (K(d) = 100 nM) than those having only two pseudo-Abeta strands. The SFAB4 protein preferentially recognized the soluble oligomeric intermediates of Abeta more than both unstructured monomer and mature amyloid fibrils. Thus, the design strategy for embedding pseudo-Abeta beta-sheet structures onto a protein surface arranged in the beta-barrel structure is useful to construct molecules capable of binding tightly to Abeta and inhibiting its aggregation. This strategy may provide implication for the diagnostic and therapeutic development in the treatment of AD.
Takahashi
Tsuyoshi
T
Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta, Yokohama 226-8501, Japan. ttakahas@bio.titech.ac.jp
Ohta
Kenichi
K
Mihara
Hisakazu
H
D000544:Alzheimer Disease / Q000188:drug therapy*; D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D055503:Protein Multimerization / Q000187:drug effects; D017433:Protein Structure, Secondary; D051381:Rats
D016229:Amyloid beta-Peptides; D049452:Green Fluorescent Proteins