Amyloid Formation by Recombinant Full-length Prion Proteins in Phospholipid Bicelle Solutions

A soluble, oligomeric β-sheet-rich conformational variant of recombinant full-length prion protein, PrPβ, was generated that aggregates into amyloid fibrils, PrPβf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrPSc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrPC to PrPβ occurs at pH 5.0 in bicellar solutions containing equimolar mixtures of dihexanoyl-phosphocholine and dimyristoyl-phospholipids, and a small percentage of negatively charged dimyristoyl-phosphoserine. The same protocol was applicable to human, cow, elk, pig, dog and mouse PrP. Comparison of full-length hPrP(23–230) with the N-terminally truncated human PrP fragments hPrP(90–230), hPrP(96–230), hPrP(105–230) and hPrP(121–230) showed that the flexible peptide segment 105–120 must be present for the generation of PrPβ. Dimerization of PrPC represents the rate-limiting step of the PrPC-to-PrPβ conformational transition, which is dependent on the amino acid sequence. The activation enthalpy of dimerization is about 130 kJ/mol for the recombinant full-length human and bovine prion proteins, and between 260 and 320 kJ/mol for the other species investigated. The in vitro conversion assay described here permits direct molecular characterization of processes that might be closely related to conformational transitions of the prion protein in transmissible spongiform encephalopathies.