|
pubmed:abstractText |
It is believed that the critical step in the pathogenesis of transmissible spongiform encephalopathies is a transition of prion protein (PrP) from an alpha-helical conformation, PrP(C), to a beta-sheet-rich form, PrP(Sc). Native prion protein contains a single disulfide bond linking Cys residues at positions 179 and 214. To elucidate the role of this bridge in the stability and folding of the protein, we studied the reduced form of the recombinant human PrP as well as the variant of PrP in which cysteines were replaced with alanine residues. At neutral pH, the reduced prion protein and the Cys-free mutant were insoluble and formed amorphous aggregates. However, the proteins could be refolded in a monomeric form under the conditions of mildly acidic pH. Spectroscopic experiments indicate that the monomeric Cys-free and reduced PrP have molten globule-like properties, i.e. they are characterized by compromised tertiary interactions, an increased exposure of hydrophobic surfaces, lack of cooperative unfolding transition in urea, and partial loss of native (alpha-helical) secondary structure. In the presence of sodium chloride, these partially unfolded proteins undergo a transition to a beta-sheet-rich structure. However, this transition is invariably associated with protein oligomerization. The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is rich in beta-sheet structure.
|
