A Disulfide-Stabilized Aβ that Forms Dimers but Does Not Form Fibrils
| Autor: | Sheng Zhang, Stan Yoo, Dalton T. Snyder, Benjamin B. Katz, Amy Henrickson, Borries Demeler, Vicki H. Wysocki, Adam G. Kreutzer, James S. Nowick |
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| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Biochemistry |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Aβ dimers are a basic building block of many larger Aβ oligomers and are among the most neurotoxic and pathologically relevant species in Alzheimer’s disease. Homogeneous Aβ dimers are difficult to prepare, characterize and study, because Aβ forms heterogeneous mixtures of oligomers that vary in size and can rapidly aggregate into more stable fibrils. This paper introduces Aβ(C18C33) as a disulfide-stabilized analogue of Aβ(42) that forms stable homogeneous dimers in lipid environments but does not aggregate to form insoluble fibrils. The Aβ(C18C33) peptide is readily expressed in E. coli and purified by reverse-phase HPLC to give ca. 8 mg of pure peptide per liter of bacterial culture. SDS-PAGE establishes that Aβ(C18C33) forms homogeneous dimers in the membrane-like environment of SDS and that conformational stabilization of the peptide with a disulfide bond prevents the formation of heterogeneous mixtures of oligomers. Mass spectrometric studies in the presence of dodecyl maltoside (DDM) further confirm the formation of stable noncovalent dimers. Circular dichroism (CD) spectroscopy establishes that Aβ(C18C33) adopts a β-sheet conformation in detergent solutions and supports a model in which the intramolecular disulfide bond induces β-hairpin folding and dimer formation in lipid environments. Thioflavin T fluorescence assays and transmission electron microscopy (TEM) studies indicate that Aβ(C18C33) does not undergo fibril formation in aqueous buffer solutions and demonstrate that the intramolecular disulfide bond prevents fibril formation. The recently published NMR structure of an Aβ(42) tetramer (PDB 6RHY) provides a working model for the Aβ(C18C33) dimer, in which two β-hairpins assemble through hydrogen bonding to form a four-stranded antiparallel β-sheet. It is anticipated that Aβ(C18C33) will serve as a stable, nonfibrilizing, noncovalent Aβ dimer model for amyloid and Alzheimer’s disease research. |
| Databáze: | OpenAIRE |
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