Two-site binding of β-cyclodextrin to the Alzheimer Aβ(1-40) peptide measured with combined PFG-NMR diffusion and induced chemical shifts

The interactions of Alzheimer's amyloid beta-peptide with cyclodextrins were studied by H-1 NMR: the translational diffusion coefficient of the peptide and chemical shift changes were studied by the presence of variable concentrations of cyclodextrins. For the full-length peptide, Abeta(1-40), the combined results of translational diffusion and chemical shift changes are consistent with a model where aromatic side chains interact with beta-cyclodextrin with dissociation constants in the millimolar range. The diffusion data were consistent with two beta-cyclodextrin molecules bound per peptide. The binding occurs at two sites, at F-19 and/or F-20 and at Y-10, with dissociation constants K-d(F) = 4.7 mM and K-d(Y) = 6.6 mM, respectively, in 10 mM sodium phosphate, pH 7.4 and 298 K. Shorter Alzheimer peptide fragments were studied to measure specific affinities for different binding sites. The N-terminal fragment Abeta(1-9) with a putative binding site at F-4 does not show measurable affinity for beta-cyclodextrin. The fragment Abeta(12-28) has similar apparent affinity (K-d = 3.8 mM) to beta-cyclodextrin as the full-length peptide Abeta(1-40). Here, the diffusion data suggests a one-to-one stoichiometry, and the binding site is F-19 and/or F-20. Both diffusion results and chemical shift changes give the same affinity. A variant Abeta(12-28)G(19)G(20) without phenylalanines does not bind to beta-cyclodextrin. Other potential ligands, alpha-cyclodextrin, gamma-cyclodextrin, nicotine, and nornicotine do not bind to the Abeta(12-28) fragment. This study shows that combined H-1 NMR diffusion and chemical shift changes may be used to quantitatively determine affinities and stoichiometries of weak interactions, using unlabeled ligands and hosts of comparable sizes.