Oxidative folding of murine prion mPrP(23-231).
A systematic study of the oxidative folding of murine prion protein mPrP(23-231) is reported here. Folding of mPrP(23-231) involves formation of a single disulfide bond, Cys179-Cys214. Despite this simplicity, reduced mPrP(23-231) exhibits numerous unusual folding properties. In the absence of denaturant, folding of mPrP(23-231) is extremely sluggish, regardless of pH. The optimal pH for mPrP(23-231) folding was found to be 4-5. At pH 8.0, a condition that typically favors disulfide formation, folding of mPrP(23-231) hardly occurs, and it not facilitated by inclusion of redox agent. In the presence of denaturant (4 M urea or 2 M guanidine hydrochloride) and basic pH (8.0), reduced mPrP(23-231) refolds to the native structure quantitatively. The efficiency of folding can be further promoted by the presence of oxidized glutathione. At pH 4.0 and in the presence of 4 M urea, reduced mPrP(23-231) converts to three distinctive conformational isomers, unable to form the native structure. These unusual properties lead us to the following conclusions. The reduced mPrP(23-231) adopts a highly rigid structure with the two cysteines buried or situated apart. The presence of denaturant or low pH disrupts this rigid structure and lowers the energy barrier, which permits oxidation and refolding of the reduced mPrP(23-231). Under selected conditions, reduced mPrP(23-231) is capable of taking on multiple forms of stable conformational isomer that are segregated by energy barriers.
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Research Center for Protein Chemistry, Institute of Molecular Medicine, University of Texas at Houston, Texas, USA.
Beck
P J
PJ
Chang
J Y
JY
D000818:Animals; D002942:Circular Dichroism; D004220:Disulfides; D019791:Guanidine; D008027:Light; D051379:Mice; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D011328:Prions / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012542:Scattering, Radiation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014508:Urea
D004220:Disulfides; D010446:Peptide Fragments; D011328:Prions; D011994:Recombinant Proteins; C428437:prion protein (23-231); D014508:Urea; D019791:Guanidine