On the Mechanism of α-Helix to β-Sheet Transition in the Recombinant Prion Protein

Abstract

It is believed that the critical event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of the prion protein from an alpha-helical form, PrP(C), to a beta-sheet-rich conformer, PrP(Sc). Recently, we have shown that incubation of the recombinant prion protein under mildly acidic conditions (pH 5 or below) in the presence of low concentrations of guanidine hydrochloride results in a transition to PrP(Sc)-like beta-sheet-rich oligomers that show fibrillar morphology and an increased resistance to proteinase K digestion [Swietnicki, W., Morillas, M, Chen, S., Gambetti, P., and Surewicz, W. K. (2000) Biochemistry 39, 424-431]. To gain insight into the mechanism of this transition, in the present study we have characterized the biophysical properties of the recombinant human prion protein (huPrP) at acidic pH in the presence of urea and salt. Urea alone induces unfolding of the protein but does not result in protein self-association or a conversion to beta-sheet structure. However, a time-dependent transition to beta-sheet structure occurs upon addition of both urea and NaCl to huPrP, even at a sodium chloride concentration as low as 50 mM. This transition occurs concomitantly with oligomerization of the protein. At a given protein and sodium chloride concentration, the rate of monomeric alpha-helix to oligomeric beta-sheet transition is strongly dependent on the concentration of urea. Low and medium concentrations of the denaturant accelerate the reaction, whereas strongly unfolding conditions are not conducive to the conversion of huPrP into an oligomeric beta-sheet-rich structure. The present data strongly suggest that partially unfolded intermediates may be involved in the transition of the monomeric recombinant prion protein into the oligomeric scrapie-like form.

Formation of Soluble Oligomers and Amyloid Fibrils with Physical Properties of the Scrapie Isoform of the Prion Protein from the C-terminal Domain of Recombinant Murine Prion Protein mPrP-(121–231)

“…In this regard, several groups have studied the conformational transition of different recombinant prion protein constructs into ␤-rich aggregates (40,(42)(43)(44)(45)(46)(47)(48). Some studies pointed to the formation of ␤-rich oligomers during the conformational transition of PrP (40,48).…”

Circular Dichroism and Its Application to the Study of Biomolecules

“…Such changes are readily studied using CD. For example, CD has been extensively used in the study of conformational transitions in peptides (see Figure 3) and in studies of the α-helix to β-sheet conversion in recombinant prion proteins induced by the addition of low concentrations of denaturants and/or salt at low pH (Morillas et al, 2001;Swietnicki et al, 2000).…”

Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

“…23 The third well characterized form of PrP is a soluble oligomeric species favored at low pH ($4) under mild denaturing conditions with a high salt content. [24][25][26][27][28] CD indicates this low pH oligomer has a high b-sheet content, and solution NMR studies have shown that the N-terminal tail retains its mobility in this oligomeric form. 28 …”