NR AXAS
AU Lu,B.Y.; Chang,J.Y.
TI A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization
QU Archives of Biochemistry and Biophysics 2007 Apr 1; 460(1): 75-84
PT journal article; research support, non-u.s. gov't
AB 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.
MH Animals; Circular Dichroism; Disulfides/*chemistry/metabolism; Guanidine/pharmacology; Hydrogen-Ion Concentration; Isomerism; Mice; Models, Molecular; Mutation; Oxidation-Reduction; Prions/*chemistry/*genetics/metabolism; Protein Conformation; Protein Denaturation; Protein Folding; Recombinant Proteins/chemistry/genetics/metabolism
AD 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.
SP englisch
PO USA