title: Disruption of prion rods generates 10-nm spherical particles having high alpha-helical content and lacking scrapie infectivity.
abstract: An abnormal isoform of the prion protein (PrP) designated PrPSc is the major, or possibly the only, component of infectious prions. Structural studies of PrPSc have been impeded by its lack of solubility under conditions in which infectivity is retained. Among the many detergents examined, only treatment with the ionic detergent sodium dodecyl sulfate (SDS) or Sarkosyl followed by sonication dispersed prion rods which are composed of PrP 27-30, an N-terminally truncated form of PrPSc. After ultracentrifugation at 100,000 x g for 1 h, approximately 30% of the PrP 27-30 and scrapie infectivity were found in the supernatant, which was fractionated by sedimentation through 5 to 20% sucrose gradients. Near the top of the gradient, spherical particles with an observed sedimentation coefficient of approximately 6S, approximately 10 mm in diameter and composed of four to six PrP 27-30 molecules, were found. The spheres could be digested with proteinase K and exhibited little, if any, scrapie infectivity. When the prion rods were disrupted in SDS and the entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods and heterogeneous particles containing high levels of prion infectivity was found. Fractions adjacent to the meniscus also contained spherical particles. Circular dichroism of the spheres revealed 60% alpha-helical content; addition of 25% acetonitrile induced aggregates high in beta sheet but remaining devoid of infectivity. Although the highly purified spherical oligomers of PrP 27-30 lack infectivity, they may provide an excellent substrate for determining conditions of renaturation under which prion particles regain infectivity.
authors.affiliation: Institut für Physikalische Biologie und Biologisch-Medizinisches Forschungszentrum, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: D
authors.initials: D
authors.lastname: Riesner
authors.forename: K
authors.initials: K
authors.lastname: Kellings
authors.forename: K
authors.initials: K
authors.lastname: Post
authors.forename: H
authors.initials: H
authors.lastname: Wille
authors.forename: H
authors.initials: H
authors.lastname: Serban
authors.forename: D
authors.initials: D
authors.lastname: Groth
authors.forename: M A
authors.initials: MA
authors.lastname: Baldwin
authors.forename: S B
authors.initials: SB
authors.lastname: Prusiner
mesh_terms: D000818:Animals; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D008647:Mesocricetus; D011328:Prions / Q000737:chemistry* / Q000302:isolation & purification / Q000472:pathogenicity* / Q000648:ultrastructure; D017433:Protein Structure, Secondary; D012608:Scrapie / Q000209:etiology*
chemical_list: D011328:Prions