Soluble amyloid A beta-(1-40) exists as a stable dimer at low concentrations

Recent studies have implicated the amyloid A beta 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 A beta or its oligomeric state, and the existing data are often contradictory, In this study, we used intrinsic fluorescence of wild type A beta-(1-40), fluorescence resonance energy transfer (FRET), and gel filtration chromatography to examine the structure of A beta-(1-40) in solution, We synthesized a series of mono-substituted fluorescent A beta-(1-40) derivatives to use as donors and accepters in FRET experiments, We selected fluorescent peptides that exhibit aggregation properties comparable to wild type A beta 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 A beta-(1-40). Equilibrium studies of the denaturation of A beta-(1-40) by increasing concentrations of dimethyl sulfoxide (Me2SO) 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% Me2SO. A beta-(1-40) is well solvated and largely monomeric in Me2SO as evidenced by a lack of FRET, When donor and acceptor AP derivatives are mixed together in Me2SO 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 A beta-(1-40), demonstrating that the fluorescent peptides interact with wild type A beta-(1-40) to form heterodimers that do not exhibit FRET, The A beta-(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 C-14-labeled A beta-(1-40) and fluorescein-labeled A beta-(1-40) elute at the same dimeric position as wild type A beta-(1-40), suggesting that soluble A beta-(1-40) is also dimeric at more physiologically plausible concentrations.