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

Bao-Yuan Lu1, Jui-Yoa Chang

  • 1Research Center for Protein Chemistry, Brown Foundation Institute of Molecular Medicine, Department of Biochemistry and Molecular Biology, The University of Texas, Houston, TX 77030, USA.

Archives of Biochemistry and Biophysics
|February 27, 2007
PubMed

Insights

Researchers engineered a mouse prion protein (mPrP) mutant to study domain interactions during oxidative folding. The resulting N-mPrP(3SS) isomer covalently links N- and C-domains, showing reduced aggregation and enabling further structural analysis.

Area of Science:

  • Biochemistry
  • Structural Biology
  • Protein Chemistry

Background:

  • Mouse prion protein (mPrP) has a disordered N-terminal domain and a structured C-terminal domain.
  • Disulfide bonds stabilize the C-terminal domain of mPrP.
  • Understanding mPrP folding and stabilization is crucial for prion disease research.

Purpose of the Study:

  • To investigate the interaction between the N- and C-domains of mPrP during oxidative folding.
  • To generate stabilized mPrP isomers for subsequent structural and functional studies.

Main Methods:

  • Expression of a mutant mPrP with four Ala/Ser to Cys replacements.
  • Oxidative folding of the reduced mutant mPrP(6C).
  • Characterization of the predominant 3-disulfide isomer (N-mPrP(3SS)) using CD spectroscopy and conformational stability assays.

Main Results:

  • Oxidative folding yielded a predominant 3-disulfide isomer, N-mPrP(3SS), linking N- and C-domains via non-native disulfide bonds.
  • N-mPrP(3SS) displayed similar CD spectra and conformational stability to wild-type mPrP.
  • The N-mPrP(3SS) isomer exhibited a reduced propensity for aggregation compared to wild-type mPrP.
  • N-mPrP(3SS) could be unfolded to generate diverse isomers for further analysis.

Conclusions:

  • Engineered disulfide bonds can covalently link the N- and C-domains of mPrP.
  • The N-mPrP(3SS) isomer offers a stable, less aggregation-prone form of mPrP.
  • This approach facilitates the isolation and analysis of various mPrP isomers.

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