Thermodynamic Investigations into Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loadings

The prion protein (PrP) is a naturally occurring protein found at high levels in central nervous system (CNS). The misfolding of the PrP is responsible for neurodegenerative diseases called transmissible spongioform encephalopathies (TSE) that include mad cow disease, scrapie in sheep and goats, kuru and Creutzfelt-Jakob disease (CJD) in humans. The normal function of the PrP is still unknown but demonstrates high selectivity for copper (Cu[superscript]+2). The mature form of PrP consists of a highly unstructured N-terminal region (23-124). The copper binding region spans from residues 60 to 96 and contains four octarepeat segments, PHGGGWGQ, and a GGGTH segment. When fully copper loaded, each octarepeat binds to a copper and the fifth copper binding involves the GGGTH site. Although the molecular details of a fully Cu[superscript]+2 loaded state are well understood, very little is known about the low copper binding state of PrP. At low Cu[superscript]+2 occupancy there is a possibility of PrP cross-linking. This project aims at obtaining the thermodynamic profile of the prion copper complex at low copper loading state to determine the forces that drive the complex formation. The model peptides were generated using solid phase peptide synthesis; the thermodynamic studies were done using isothermal titration calorimetry (ITC) and supporting spectroscopic studies by circular dichroism (CD). Examination of the ITC titration data suggests an initial binding event where two PrP's are cross-linked by a single copper ion. ITC titrations were performed in both forward and reverse directions in order to examine the reversibility of the copper binding process. Fitting the ITC data with the existing models, i.e one sets of sites, two sets of sites or sequential binding, lead to unsatisfactory fits suggesting a more complex binding process. The hypothesized binding model will hopefully lead to good fits for the ITC data and will support the hypothesis that Cu[superscript]+2 is cross- linking PrP molecules at low equivalents of added Cu[superscript]+2 .  M.S

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

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., Δ<i>H</i> and Δ<i>S</i>) 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 (<i>K</i>, Δ<i>G</i>, Δ<i>H</i>, and <i>T</i>Δ<i>S</i>) 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 <i>N</i>-(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 <i>K</i>_d 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

Thermodynamic Investigations into Copper Binding in the N-Terminal Region of the Prion Protein at Low Copper Loadings

Abstract  Thermodynamic Investigation into Copper Binding  in the N-Terminal Region of the Prion Protein at Low Copper Loadings  By  Devi Praneetha Gogineni  December 2010  Chair: Rickey Hicks, Ph.D.  Major Department: Chemistry  The prion protein (PrP) is a naturally occurring protein found at high levels in central nervous system (CNS). The misfolding of the PrP is responsible for neurodegenerative diseases called transmissible spongioform encephalopathies (TSE) that include mad cow disease, scrapie in sheep and goats, kuru and Creutzfelt-Jakob disease (CJD) in humans. The normal function of the PrP is still unknown but demonstrates high selectivity for copper (Cu+2). The mature form of PrP consists of a highly unstructured N-terminal region (23-124). The copper binding region spans from residues 60 to 96 and contains four octarepeat segments, PHGGGWGQ, and a GGGTH segment. When fully copper loaded, each octarepeat binds to a copper and the fifth copper binding involves the GGGTH site. Although the molecular details of a fully Cu+2 loaded state are well understood, very little is known about the low copper binding state of PrP. At low Cu+2occupancy there is a possibility of PrP cross-linking. This project aims at obtaining the thermodynamic profile of the prion copper complex at low copper loading state to determine the forces that drive the complex formation. The model peptides were generated using solid phase peptide synthesis; the thermodynamic studies were done using isothermal titration calorimetry (ITC) and supporting spectroscopic studies by circular dichroism (CD). Examination of the ITC titration data suggests an initial binding event where two PrP's are cross-linked by a single copper ion. ITC titrations were performed in both forward and reverse directions in order to examine the reversibility of the copper binding process. Fitting the ITC data with the existing models, i.e one sets of sites, two sets of sites or sequential binding, lead to unsatisfactory fits suggesting a more complex binding process. The hypothesized binding model will hopefully lead to good fits for the ITC data and will support the hypothesis that Cu+2 is cross- linking PrP molecules at low equivalents of added Cu+2 .