The role of disulfide bridge in the folding and stability of the recombinant human prion protein

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, PrPC, to a beta -sheet-rich form, PrPSc. Nativ e prion protein contains a single disulfide bond linking Cys residues at po sitions 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 wi th alanine residues. At neutral pH, the reduced prion protein and the Cys-f ree mutant were insoluble and formed amorphous aggregates. However, the pro teins could be refolded in a monomeric form under the conditions of mildly acidic pH, Spectroscopic experiments indicate that the monomeric Cys-free a nd reduced PrP have molten globule-like properties, i.e. they are character ized by compromised tertiary interactions, an increased exposure of hydroph obic surfaces, lack of cooperative unfolding transition in urea, and partia l loss of native (alpha -helical) secondary structure. In the presence of s odium chloride, these partially unfolded proteins undergo a transition to a beta -sheet-rich structure. However, this transition is invariably associa ted with protein oligomerization, The present data argue against the notion that reduced prion protein can exist in a stable monomeric form that is ri ch in beta -sheet structure.