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Soluble Amyloid Aβ-(1–40) Exists as a Stable Dimer at Low Concentrations

1997; Elsevier BV; Volume: 272; Issue: 34 Linguagem: Inglês

10.1074/jbc.272.34.21037

1083-351X

William Garzon-Rodriguez, Marisa Sepulveda-Becerra, Saskia Milton, Charles Glabe,

Computational Drug Discovery Methods

Recent studies have implicated the amyloid Aβ peptide and its ability to self-assemble as key factors in the pathogenesis of Alzheimer's disease. Relatively little is known about the structure of soluble Aβ or its oligomeric state, and the existing data are often contradictory. In this study, we used intrinsic fluorescence of wild type Aβ-(1–40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of Aβ-(1–40) in solution. We synthesized a series of mono-substituted fluorescent Aβ-(1–40) derivatives to use as donors and acceptors in FRET experiments. We selected fluorescent peptides that exhibit aggregation properties comparable to wild type Aβ for analysis in donor-acceptor pairs; two labeled with 5-(2-((iodoacetyl)amino)ethyl)aminonaphthylene-1-sulfonic acid at Cys-25 or Cys-34 and fluorescein maleimide at Cys-4 or Cys-7. Another peptide containing a Trp substitution at position 10 was used as an acceptor for the intrinsic Tyr fluorescence of wild type Aβ-(1–40). Equilibrium studies of the denaturation of Aβ-(1–40) by increasing concentrations of dimethyl sulfoxide (Me 2 SO) were conducted by monitoring fluorescence, with a midpoint value for the unfolding transition of both the substituted and wild type peptides at among 40 and 50% Me 2 SO. Aβ-(1–40) is well solvated and largely monomeric in Me 2 SO as evidenced by a lack of FRET. When donor and acceptor Aβ derivatives are mixed together in Me 2 SO and then diluted 10-fold into aqueous Tris-HCl buffer at pH 7.4, efficient FRET is observed immediately for all pairs of fluorescent peptides, indicating that donor-acceptor dimers exist in solution. FRET is abolished by the addition of an excess of unlabeled Aβ-(1–40), demonstrating that the fluorescent peptides interact with wild type Aβ-(1–40) to form heterodimers that do not exhibit FRET. The Aβ-(1–40) dimers appear to be very stable, because no subunit exchange is observed after 24 h between fluorescent homodimers. Gel filtration confirms that nanomolar concentrations of 14 C-labeled Aβ-(1–40) and fluorescein-labeled Aβ-(1–40) elute at the same dimeric position as wild type Aβ-(1–40), suggesting that soluble Aβ-(1–40) is also dimeric at more physiologically plausible concentrations.