Abstract
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.
Highlights
Mammalian prion diseases, known as transmissible spongiform encephalopathies, are a group of fatal neurodegenerative disorders that include scrapie in sheep and goat, bovine spongiform encephalopathy (BSE)1 in cattle and CreutzfeldtJakob disease in humans
We characterize two partially folded equilibrium intermediates of the murine prion protein stabilized by high hydrostatic pressure and low temperature
The equilibrium unfolding of the recombinant murine prion protein, mPrP-(121–231), was investigated using chemical and physical denaturants. mPrP-(121– 231) corresponds to the structured 111-residue domain of the cellular prion protein, and its structure is the same as that in the context of the full-length protein [7, 10]
Summary
Chemicals—bis-ANS was from Molecular Probes (Eugene, OR). Stock solutions were prepared in methanol, and the concentration was determined using the extinction coefficient, ⑀360 ϭ 23,000 MϪ1 cmϪ1 [33]. Bis-ANS fluorescence was excited at 365 nm, and emission spectra were recorded from 420 to 600 nm. Thermodynamic Parameters of Unfolding of mPrP[121–231]—⌬GuH2O (the free energy of unfolding in the absence of denaturant) and the m value, which measures the steepness of the dependence of the unfolding transition on the concentration of denaturant, were obtained from urea unfolding data using the linear extrapolation method [36]. 1. Equilibrium unfolding of F175W mPrP-(121–231) by urea monitored by intrinsic (f) and bis-ANS (G) fluorescence. Fluorescence measurements were carried out as described under “Experimental Procedures.” bis-ANS and intrinsic fluorescence intensities are normalized by their respective values in the absence of urea. The temperature dependence of the equilibrium constant for a two-state unfolding transition is described by the van’t. From a plot of ⌬G/T versus the inverse temperature, the changes in enthalpy (⌬H) and entropy (⌬S) of unfolding can be extracted
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