title: Physical studies of conformational plasticity in a recombinant prion protein.
abstract: PrP(Sc) is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrP(Sc) produces an N-terminally truncated polypeptide of about 142 residues, designated PrP 27-30. Recently, a recombinant protein (rPrP) of 142 residues corresponding to the Syrian hamster PrP 27-30 was expressed in Escherichia coli and purified (Mehlhorn et al., 1996). rPrP has been refolded into both alpha-helical and beta-sheet structures as well as various intermediates in aqueous buffers. The beta-sheet state and two pH-dependent alpha-helical states were characterized by CD and NMR. The alpha-helical conformation occurred only after the formation of an intramolecular disulfide bond, whereas the beta-sheet form was accessible either with or without the disulfide. Of the different alpha-helical forms studied, only those refolded in the pH range 5-8 were substantially soluble at physiological pH, exhibiting similar conformations and monomeric analytical sedimentation profiles throughout the above pH range. Furthermore, refolded alpha-rPrP showed NMR chemical shift dispersion typical of proteins with native conformations, although 2D NMR indicated large segments of conformational flexibility. It displayed a cooperative thermal denaturation transition; at elevated temperatures, it converted rapidly and irreversibly to the thermodynamically more stable beta-sheet form. Unfolding of alpha-rPrP by GdnHCl revealed a two-phase transition with a relatively stable folding intermediate at 2 M GdnHCl. The deltaG values were estimated to be 1.9 +/- 0.4 kcal/mol for the first phase and 6.5 +/- 1.2 kcal/mol for the second, consistent with a folding core surrounded by significant segments of flexible conformation. By NMR, alpha-rPrP(acid) isolated at pH 2 without refolding exhibited heterogeneous line widths, consistent with an acid-denatured molten globular state. We conclude that to the extent that rPrP constitutes a relevant folding domain of PrP(C), the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrP(C) may possess a relatively open conformation which makes it susceptible to conversion into PrP(Sc) under appropriate conditions.
authors.affiliation: Department of Neurology, University of California, San Francisco 94143, USA.
authors.forename: H
authors.initials: H
authors.lastname: Zhang
authors.forename: J
authors.initials: J
authors.lastname: Stockel
authors.forename: I
authors.initials: I
authors.lastname: Mehlhorn
authors.forename: D
authors.initials: D
authors.lastname: Groth
authors.forename: M A
authors.initials: MA
authors.lastname: Baldwin
authors.forename: S B
authors.initials: SB
authors.lastname: Prusiner
authors.forename: T L
authors.initials: TL
authors.lastname: James
authors.forename: F E
authors.initials: FE
authors.lastname: Cohen
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D006224:Cricetinae; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008647:Mesocricetus; D018583:Pliability; D011328:Prions / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared; D014461:Ultracentrifugation
chemical_list: D011328:Prions; D011994:Recombinant Proteins