Amyloid formation by recombinant full-length prion proteins in phospholipid bicelle solutions.
A soluble, oligomeric beta-sheet-rich conformational variant of recombinant full-length prion protein, PrP beta, was generated that aggregates into amyloid fibrils, PrP betaf. These fibrils have physico-chemical and structural properties closely similar to those of pathogenic PrP Sc in scrapie-associated fibrils and prion rods, including a closely similar proteinase K digestion pattern and Congo red birefringence. The conformational transition from PrP C to PrP beta 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 PrP beta. Dimerization of PrP C represents the rate-limiting step of the PrP C-to-PrP beta 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.
Lührs
Thorsten
T
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Zürich, CH-8093 Zürich, Switzerland.
Zahn
Ralph
R
Wüthrich
Kurt
K
D000682:Amyloid / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003670:Deer; D004285:Dogs; D006801:Humans; D007700:Kinetics; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism*; D051379:Mice; D046529:Microscopy, Electron, Transmission; D010743:Phospholipids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011328:Prions / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D012996:Solutions; D013552:Swine
D000682:Amyloid; D008051:Lipid Bilayers; D010743:Phospholipids; D011328:Prions; D011994:Recombinant Proteins; D012996:Solutions