Abstract
Transmissible spongiform encephalopathies are associated with the conversion of cellular prion protein, PrP(C), into a misfolded oligomeric form, PrP(Sc). Here we have examined the kinetics of folding and unfolding reactions for the recombinant human prion protein C-terminal fragment 90-231 at pH 4.8 and 7.0. The stopped-flow data provide clear evidence for the population of an intermediate on the refolding pathway of the prion protein as indicated by a pronounced curvature in chevron plots and the presence of significant burst phase amplitude in the refolding kinetics. In addition to its role in the normal prion protein folding, this intermediate likely represents a crucial monomeric precursor of the pathogenic PrP(Sc) isoform.
Highlights
From the Department of Physiology and Biophysics and Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106
Folding Intermediate of Prion Protein ground, substituting Phe or Tyr at different positions with tryptophan. Preliminary characterization of these single Trp variants has identified the Y218W mutant (Fig. 1) as potentially the most suitable candidate for stopped-flow experiments. Both at neutral and acidic pH, the Y218W substitution is essentially nonperturbing as indicated by far-UV circular dichroism spectra and equilibrium urea unfolding curves
Within the context of the protein-only hypothesis, the key event in the pathogenesis of prion diseases is the conversion of prion protein from ␣-helical form, PrPC, to a -sheet-rich conformer, PrPSc [1]
Summary
PrPC, cellular prion protein; PrP, prion protein; PrPSc, pathogenic PrP isoform. swapped dimer [6]. PrPC, cellular prion protein; PrP, prion protein; PrPSc, pathogenic PrP isoform. This dimer, which is only marginally populated in solution and selectively crystallizes, is ␣-helical, and its overall fold is similar to that of the monomer. In contrast to a largely ␣-helical PrPC, the pathogenic PrPSc isoform is characterized by a high content of -sheet structure [7, 8], partial resistance to proteolytic digestion, and a propensity to aggregate into insoluble amyloid-like fibrils and plaques [1]. We present evidence for a population of a kinetic intermediate during the folding of the human prion protein. This species may represent a crucial monomeric precursor on the pathway of prion protein conversion to the pathogenic PrPSc isoform
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