A survey of diamagnetic probes for copper2+ binding to the prion protein. 1H NMR solution structure of the palladium2+ bound single octarepeat.

The prion protein (PrP(C)) is a copper binding cell surface glycoprotein which when misfolded causes transmissible spongiform encephalopathies. The cooperative binding of Cu2+ to an unstructured octarepeat sequence within PrP(C) causes profound folding of this region. The use of NMR to determine the solution structure of the octarepeat region of PrP with Cu2+ bound has been hampered by the paramagnetic nature of the Cu2+ ions. Using NMR we have investigated the binding of candidate diamagnetic replacement ions, to the octarepeat region of PrP. We show that Pd2+ forms diamagnetic complexes with the peptides HGGG, HGGGW and QPHGGGWGQ with 1:1 stoichiometry. The 1H NMR spectra indicate that these peptides are in slow-exchange between free and bound Pd2+ on the chemical-shift time-scale. We demonstrate that the Pd-peptide complex forms slowly with a time taken to reach half-maximal signal of 3 hours. Other candidate metal ions, Ni2+, Pt2+ and Au3+, were investigated but only the Pd2+ complexes gave resolvable 1H NMR spectra. We have determined the solution structure of the QPHGGGWGQ-Pd 1:1 complex using 71 NOE distance restraints. A backbone RMSD of 0.30 A was observed over residues 3 to 7 in the final ensemble. The co-ordinating ligands consist of the histidine imidazole side chain N epsilon, the amide N of the second and third glycines with possibly H2O as the fourth ligand. The co-ordination geometry differs markedly from that of the HGGGW-Cu crystal structure. This survey of potential replacement metal ions to Cu2+ provides insight into the metal specificity and co-ordination chemistry of the metal bound octarepeats.