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 (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) 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 (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) 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.
Lu
Bao-Yuan
BY
Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, USA.
Chang
Jui-Yoa
JY
D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007536:Isomerism; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D010084:Oxidation-Reduction; D000072002:Prion Proteins; D011328:Prions / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D004220:Disulfides; D000072002:Prion Proteins; D011328:Prions; C512657:Prnp protein, mouse; D011994:Recombinant Proteins; D019791:Guanidine