A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23–231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23–124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys178–Cys213). We have expressed a mPrP mutant with 4 Ala/Ser ⇒ Cys replacements, two each at the N-(Cys36, Cys112) and C-(Cys134, Cys169) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys178–Cys213) and two non-native disulfide bonds (Cys36–Cys134 and Cys112–Cys169) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23–231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.