| Abstract
| - The infectious isoform of the prion protein(PrPSc) is derived from cellular PrP (PrPC) inaconversion reaction involving a dramatic reorganization of secondaryand tertiary structure. While ourunderstanding of the pathogenic role of PrPSc has grown,the normal physiologic function of PrPC stillremains unclear. Using recombinant Syrian hamster prion protein[SHaPrP(29−231)], we investigatedmetal ions as possible ligands of PrP. Near-UV circular dichroismspectroscopy (CD) indicates that theconformation of SHaPrP(29−231) resembles PrPCpurified from hamster brain. Here we demonstrate byCD and tryptophan (Trp) fluorescence spectroscopy that copper induceschanges to the tertiary structureof SHaPrP(29−231). Binding of copper quenches the Trpfluorescence emission significantly, shifts theemission spectrum to shorter wavelengths, and also induces changes inthe near-UV CD spectrum ofSHaPrP(29−231). The binding sites are highly specific forCu2+, as indicated by the lack of a changeinTrp fluorescence emission with Ca2+, Co2+,Mg2+, Mn2+, Ni2+, andZn2+. Binding of Cu2+ alsopromotesthe conformational shift from a predominantly α-helical to aβ-sheet structure. Equilibrium dialysisexperiments indicate a binding stoichiometry of ∼2 copper moleculesper PrP molecule at physiologicallyrelevant concentrations, and pH titration of Cu2+ bindingsuggests a role for histidine as a chelating ligand.NMR spectroscopy has recently demonstrated that the octarepeats(PHGGGWGQ) in SHaPrP(29−231)lack secondary or tertiary structure in the absence ofCu2+. Our results suggest that eachCu2+ binds toa structure defined by two octarepeats (PHGGGWGQ) with one histidineand perhaps one glycine carbonylchelating the ion. We propose that the binding of two copper ionsto four octarepeats induces a moredefined structure to this region.
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