Quantification of the Binding Constant of Copper(II) to the Amyloid-Beta Peptide

Abstract

The amyloid beta (A beta) peptide of Alzheimer's disease binds copper(II), and the peptide-bound metal may be a source of reactive oxygen species and neurotoxicity. To circumvent peptide aggregation and reduce redox activity, there is growing interest in using metal chelates as drug therapeutics for AD, whose design requires accurate data on the affinity of A beta peptides for copper(II). Reports on Cu2+ binding to A beta range from approximately 10(5) to approximately 10(9); these values' being obtained for different peptide lengths (1-16, 1-28, 1-40, 1-42) at varying pH. Herein, we report that Cu2+'s binding to A beta(1-40) at 37 degrees C occurs in a 1:1 stoichiometry with a pH-dependent binding constant: 1.1 (+/-0.2) x 10 (9) M (-1) and 2.4 (+/-0.2) x 10 (9) M(-1) at pH 7.2 and 7.4, respectively. Under identical conditions, A beta(1-16) reveals a comparable binding constant, confirming that this portion of the peptide is the binding region. Several previously reported values can be reconciled with the current measurement by careful consideration of thermodynamics associated with the presence of competing ligands used to solubilize copper.

Thermodynamic study of Cu2+ binding to the DAHK and GHK peptides by isothermal titration calorimetry (ITC) with the weaker competitor glycine

“…A weaker ligand is necessary in order to measure the otherwise too strong Cu 2+ binding [23]. We were able to show that these ITC measurements were in agreement with the structural and thermodynamic values from the literature, suggesting that the here applied methodology might be useful for other less well-characterized copper-binding peptides, like amyloid-β [23,24], prion, -synculein etc.…”

“…First, the Cu 2+ -binding to Aβ16 was measured under the same conditions applied by Hatcher et al [23]. This enabled the comparison of our measurement with the literature data.…”

“…Although in a 100mM HEPES buffer the affinity will decrease to a app Kd of about 10 -12 M at pH 7.4 [26], this affinity is still too high to be measured directly by ITC. Thus we applied the competitive titration method using glycine as a competitor, following the approach published to study the Cu 2+ binding to the Aβ peptide [23,24]. In the presence of the weaker competitor glycine, the apparent app Kd will be higher and hence accessible by ITC measurements.…”

“…Study of Cu 2+ binding to Aβ16 peptide: The binding of Cu 2+ to Aβ16 was studied previously using ITC [23][24][25]. Since to the scope of our work included a comparison of thermodynamic profiles of …”

Modeling Cu2+-Aβ complexes from computational approaches

“…40,41 It is worth mentioning that many experiments have also been done using the truncated Aβ (1-16) peptide, since it is more soluble than Aβ(1-42) and experimental evidences have shown that it can be very useful to understand its redox behavior, metal cation affinities, metal coordination and other physicochemical properties. 20,22,[42][43][44][45] Other truncated models such as Cu 2+ -Aβ(1-7) have been considered to study the mechanism of Cu 2+ reduction using quantum chemical methods, 37 but the interaction of Cu 2+ with larger systems including the whole Aβ(1-42) peptide have only been considered by means of classical molecular dynamic simulations. 46 The consideration of the full peptide Aβ(1-42) at the quantum mechanics level is computationally too expensive and thus, metal ion complexes are studied by using computational approaches that combine a quantum mechanical treatment for the copper coordination center and a molecular mechanics (MM) approach for the peptide moiety.…”