Multiple folding pathways for heterologously expressed human prion protein

Human PrP (residues 91-231) expressed in Escherichia coli can adopt several conformations in solution depending on pH, redox conditions and denaturant concentration. Oxidised PrP at neutral pH, with the disulphide bond intact , is a soluble monomer which contains 47% alpha-helix and corresponds to Pr PC. Denaturation studies show that this structure has a relatively small, s olvent-excluded core and unfolds to an unstructured state in a single, co-o perative transition with a Delta G for folding of -5.6 kcal mol(-1). The un folding behaviour is sensitive to pH and at 4.0 or below the molecule unfol ds via a stable folding intermediate. This equilibrium intermediate has a r educed helical content and aggregates over several hours. When the disulphi de bond is reduced the protein adopts different conformations depending upo n pH, At neutral pH or above, the reduced protein has an alpha-helical fold , which is identical to that observed for the oxidised protein. At pH 4 or below, the conformation rearranges to a fold that contains a high proportio n of beta-sheet structure. In the reduced state the alpha- and beta-forms a re slowly inter-convertible whereas when oxidised the protein can only adop t an alpha-conformation in free solution. The data we present here shows th at the human prion protein can exist in multiple conformations some of whic h are knows to be capable of forming fibrils. The precise conformation that human PrP adopts and the pathways for unfolding are dependent upon solvent conditions. The conditions we examined are within the range that a protein may encounter in sub-cellular compartments and may have implications for t he mechanism of conversion of PrPC to prp(Se) in vivo. Since the conversion of PrPC to PrPSe is accompanied by a switch in secondary structure from al pha to beta, this system provides a useful model for studying major structu ral rearrangements in the prion protein. (C) 1999 Elsevier Science B.V. All rights reserved.