Calorimetric investigation of copper binding in the N-terminal region of the prion protein at low copper loading: evidence for an entropically favorable first binding event.

Abstract

Although the Cu2+-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu2+, the Cu2+-loading mechanism is just beginning to come into view. Because the Cu2+-binding modes at low and intermediate Cu2+ occupancy necessarily represent the highest-affinity binding modes, these are very likely populated under physiological conditions, and it is thus essential to characterize them in order to understand better the biological function of copper–prion interactions. Besides binding-affinity data, almost no other thermodynamic parameters (e.g., ΔH and ΔS) have been measured, thus leaving undetermined the enthalpic and entropic factors that govern the free energy of Cu2+ binding to the prion protein. In this study, isothermal titration calorimetry (ITC) was used to quantify the thermodynamic parameters (K, ΔG, ΔH, and TΔS) of Cu2+ binding to a peptide, PrP(23–28, 57–98), that encompasses the majority of the residues implicated in Cu2+ binding by full-length PrP. Use of the buffer N-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu2+ chelator, allowed for the isolation of the two highest affinity binding events. Circular dichroism spectroscopy was used to characterize the different binding modes as a function of added Cu2+. The Kd values determined by ITC, 7 and 380 nM, are well in line with those reported by others. The first binding event benefits significantly from a positive entropy, whereas the second binding event is enthalpically driven. The thermodynamic values associated with Cu2+ binding by the Aβ peptide, which is implicated in Alzheimer’s disease, bear striking parallels to those found here for the prion protein.