Abstract
Both prion protein and the structurally homologous protein doppel are associated with neurodegenerative disease by mechanisms which remain elusive. We have prepared murine doppel, and a mutant with one of the two disulphide bonds removed, in the expectation of increasing the similarity of doppel to prion protein in terms of conformation and stability. Unfolding studies of doppel and the mutant have been performed using far-UV CD over a range of solution conditions known to favour the alpha-->beta transformation of recombinant prion protein. Only partial unfolding of doppel or the mutant occurs at elevated temperature, but both exhibit full and reversible unfolding in chemical denaturation with urea. Doppel is significantly less stable than prion protein, and this stability is further reduced by removal of the disulphide bond between residues 95-148. Both doppel and the mutant are observed to unfold by a two-state mechanism, even under the mildly acidic conditions where prion protein forms an equilibrium intermediate with enhanced beta-structure, potentially analogous to the conversion of the cellular form of the prion protein into the infectious form (PrP(C)-->PrP(Sc)). Furthermore, no direct interaction of either doppel protein with prion protein, either in the alpha-form or the beta-rich conformation, was detectable spectroscopically. These studies indicate that, in spite of the similarity in secondary structure between the doppel and prion protein, there are significant differences in their solution properties. The fact that neither doppel nor its mutant exhibited the alpha-->beta transformation of the prion protein suggests that this conversion property may be dependent on unique sequences specific to the prion protein.