NR AMZN
AU Zhang,H.; Stockel,J.; Mehlhorn,I.; Groth,D.; Baldwin,M.A.; Prusiner,S.B.; James,T.L.; Cohen,F.E.
TI Physical studies of conformational plasticity in a recombinant prion protein
QU Biochemistry 1997 Mar 25; 36(12): 3543-53
PT journal article
AB PrPsc is known to be the major, if not the only, component of the infectious prion. Limited proteolysis of PrPsc 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 PrPc, the various conformations exhibited by rPrP suggest that the PrP sequence may be intrinsically plastic in its conformations; indeed, portions of PrPc may possess a relatively open conformation which makes it susceptible to conversion into PrPsc under appropriate conditions.
MH Animal; Chromatography, High Pressure Liquid; Circular Dichroism; Hamsters; Magnetic Resonance Spectroscopy; Mesocricetus; Pliability; Prions/*chemistry; Protein Conformation; Recombinant Proteins/*chemistry; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Mass; Support, Non-U.S. Gov't; Support, U.S. Gov't, P.H.S.; Ultracentrifugation
AD Department of Neurology, University of California, San Francisco 94143, USA
SP englisch
PO USA