Folding intermediates of the prion protein stabilized by hydrostatic pressure and low temperature.

Prion diseases are associated with conformational conversion of the cellular prion protein, PrPC, into a misfolded form, PrPSc. We have investigated the equilibrium unfolding of the structured domain of recombinant murine prion protein, comprising residues 121-231 (mPrP-(121-231)). The equilibrium unfolding of mPrP-(121-231) by urea monitored by intrinsic fluorescence and circular dichroism (CD) spectroscopies indicated a two-state transition, without detectable folding intermediates. The fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5-disulfonic acid (bis-ANS) binds to native mPrP-(121-231), indicating exposure of hydrophobic domains on the protein surface. Increasing concentrations of urea (up to 4 M) caused the release of bound bis-ANS, whereas changes in intrinsic fluorescence and CD of mPrP took place only above 4 M urea. This indicates the existence of a partially unfolded conformation of mPrP, characterized by loss of bis-ANS binding and preservation of the overall structure of the protein, stabilized at low concentrations of urea. Hydrostatic pressure and low temperatures were also used to stabilize partially folded intermediates that are not detectable in the presence of chemical denaturants. Compression of mPrP to 3.5 kbar at 25 degrees C and pH 7 caused a slight decrease in intrinsic fluorescence emission and an 8-fold increase in bis-ANS fluorescence. Lowering the temperature to -9 degrees C under pressure reversed the decrease in intrinsic fluorescence and caused a marked (approximately 40-fold) increase in bis-ANS fluorescence. The increase in bis-ANS fluorescence at low temperatures was similar to that observed for mPrP at 1 atm at pH 4. These results suggest that pressure-assisted cold denaturation of mPrP stabilizes a partially folded intermediate that is qualitatively similar to the state obtained at acidic pH. Compression of mPrP in the presence of a subdenaturing concentration of urea stabilized another partially folded intermediate, and cold denaturation under these conditions led to complete unfolding of the protein. Possible implications of the existence of such partially folded intermediates in the folding of the prion protein and in the conversion to the PrPSc conformer are discussed.