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

Abstract

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 .