title: 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 Cu(2+)-binding sites of the prion protein have been well studied when the protein is fully saturated by Cu(2+), the Cu(2+)-loading mechanism is just beginning to come into view. Because the Cu(2+)-binding modes at low and intermediate Cu(2+) 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 Cu(2+) 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 Cu(2+) binding to a peptide, PrP(23-28, 57-98), that encompasses the majority of the residues implicated in Cu(2+) binding by full-length PrP. Use of the buffer N-(2-acetomido)-aminoethanesulfonic acid (ACES), which is also a well-characterized Cu(2+) 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 Cu(2+). 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 Cu(2+) binding by the Aβ peptide, which is implicated in Alzheimer's disease, bear striking parallels to those found here for the prion protein.
authors.affiliation: Department of Chemistry, East Carolina University , East 5th Street, Greenville, North Carolina 27858, United States.
authors.forename: Devi Praneetha
authors.initials: DP
authors.lastname: Gogineni
authors.forename: Anne M
authors.initials: AM
authors.lastname: Spuches
authors.forename: Colin S
authors.initials: CS
authors.lastname: Burns
mesh_terms: D002151:Calorimetry; D003300:Copper / Q000378:metabolism*; D019277:Entropy; D006639:Histidine; D011328:Prions / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D011328:Prions; D006639:Histidine; D003300:Copper