Calorimetric investigation of copper(II) binding to Aβ peptides: thermodynamics of coordination plasticity

Abstract

Metal ions have been shown to play a critical role in β-amyloid (Aβ) neurotoxicity, thus prompting an intense investigation into the formation of metal-Aβ complexes. Isothermal titration calorimetry (ITC) has been widely used to determine binding constants (K) for a variety of metal-protein interactions, including those in metal-Aβ complexes. In this study, ITC was used to more fully quantify the thermodynamics (K, ΔG, ΔH, and TΔS) of Cu(2+) binding to Aβ16, N-acetyl-Aβ16, Aβ28, N-acetyl-Aβ28, and Aβ28 variants (H6A, H13A, H14A) at pH 7.4 and 37 °C. After deconvolution of competing reactions, K for Aβ16 was found to be 1.1 (±0.13) × 10(9) and is in strong agreement with literature values measured under similar conditions. Further, a similar K value was obtained at two additional concentrations of competing ligand, suggesting that ternary complex formation is not significant. The acetylated peptide analogs reveal a marked decrease in the overall free energy upon binding, which is the result of less favorable enthalpic and entropic contributions. Circular dichroism spectroscopy shows conformational changes that are consistent with these results. Most importantly, data for Aβ28 variants lacking a potential Cu(2+)-binding histidine residue reveal that the overall free energy of binding remains constant, which is the result of entropy/enthalpy compensation. These data provide fundamental thermodynamic evidence for coordination plasticity in Cu(2+) binding to Aβ and other intrinsically disordered peptides.

Dissecting ITC data of metal ions binding to ligands and proteins

“…Comparing values determined in experimentally distinct conditions (e.g. varying the buffer), with the proper control experiments and calculations, may provide further support for the presence or absence of mixed species [18].…”

“…UV-vis spectroscopy and potiometry) are required [35,36]. This complication can be avoided by selecting a buffer that can maintain the desired pH as well as chelate the metal thus preventing metal hydroxide precipitation and minimizing the species in solution [18]. Since the thermodynamics of a variety of metal-buffer interactions have been well documented [37][38][39][40] or can be experimentally determined [41] as discussed below, these interactions can readily be accounted for while processing experimental data.…”

“…Alternatively, Zn 2+ binding events are characterized by a quantifiable heat signature which can be exploited by ITC to gain insight into Zn 2 + -dependent systems such as zinc finger domains [4][5][6][7], carbonic anhydrase [8][9][10], and insulin [11]. Additionally, ITC has played a role in understanding countless important bioinorganic systems including ferritin and transferrin [12][13][14][15][16][17], amyloid beta [18,19], nucleases [20][21][22][23] and cardiac troponin C [24].…”

Detection of some Heavy Metals Using Rhodamine B Isothiocyanate Fluoresceinated Dye

“…ACES was used recently to assist calorimetric determination of the affinity of Cu (II) to Alzheimer's Disease's A β peptide and some other bioligands (Grossoehme et al, 2010;Sacco et al, 2012).…”

Non-intercalative, Deoxyribose Binding of Boric Acid to Calf Thymus DNA

“…Several reports in literature detail ITC-based quantitative measurements of interactions between biological macromolecules, such as proteins [30][31][32][33][34][35] or DNA [36][37][38][39] and metal ions. In this work, ITC was utilized to characterize the thermodynamics of boric acid binding to ct-DNA.…”