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Oxidative folding of murine prion mPrP(23-231)
1Research Center for Protein Chemistry, Institute of Molecular Medicine, University of Texas at Houston, Texas, USA.
Abstract:
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.
Insights
Murine prion protein (mPrP) folding is slow and pH-dependent, requiring denaturants or low pH to facilitate disulfide bond formation and achieve native structure. This protein can form multiple stable isomers under specific conditions.
Area of Science:
- Biochemistry
- Protein Folding
- Structural Biology
Background:
- Prion proteins are implicated in neurodegenerative diseases.
- Understanding the folding mechanisms of prion proteins is crucial for disease research.
- Murine prion protein (mPrP) folding involves a single disulfide bond (Cys179-Cys214).
Purpose of the Study:
- To systematically investigate the oxidative folding properties of reduced murine prion protein mPrP(23-231).
- To elucidate the factors influencing mPrP folding kinetics and structural outcomes.
- To characterize the unusual folding behavior of mPrP under various conditions.
Main Methods:
- Systematic study of mPrP(23-231) oxidative folding under varying pH, denaturant concentrations, and redox conditions.
- Utilized urea and guanidine hydrochloride as denaturants.
- Investigated the role of oxidized glutathione as a folding promoter.
Main Results:
- Reduced mPrP(23-231) exhibits sluggish folding in the absence of denaturants, with optimal folding at pH 4-5.
- Folding is minimal at pH 8.0, even with redox agents.
- Denaturants (4 M urea or 2 M guanidine hydrochloride) combined with basic pH (8.0) promote quantitative refolding to the native structure.
- Oxidized glutathione enhances folding efficiency.
- At pH 4.0 with 4 M urea, mPrP(23-231) forms three distinct conformational isomers.
Conclusions:
- Reduced mPrP(23-231) possesses a rigid structure with buried or separated cysteines.
- Denaturants or low pH disrupt this rigidity, lowering energy barriers for oxidation and refolding.
- mPrP(23-231) can adopt multiple stable conformational isomers separated by energy barriers under specific conditions.