The prion protein is known to be a copper-binding protein, but affinity and
stoichiometry data for the full-length protein at a physiological pH of 7
were lacking. Furthermore, it was unknown whether only the highly flexible
N-terminal segment with its octarepeat region is involved in copper binding
or whether the structured C-terminal domain is also involved, Therefore we
systematically investigated the stoichiometry and affinity of copper bindi
ng to full-length prion protein PrP23-231 and to different N- and C-termina
l fragments using electrospray ionization mass spectrometry and fluorescenc
e spectroscopy. Our data indicate that the unstructured N-terminal segment
is the cooperative copper-binding domain of the prion protein. The prion pr
otein binds up to five copper(II) ions with half-maximal binding at similar
to2 muM. This argues strongly for a direct role of the prion protein in co
pper metabolism, since it is almost saturated at about 5 muM, and the excha
ngeable copper Fool concentration in blood is about 8 muM.