DISRUPTION OF PRION RODS GENERATES 10-NM SPHERICAL-PARTICLES HAVING HIGH ALPHA-HELICAL CONTENT AND LACKING SCRAPIE INFECTIVITY

An abnormal isoform of the prion protein (PrP) designated PrPSe is the major, or possibly the only, component of infectious prions, Structur al studies of PrPSe have been impeded by its lack of solubility under conditions in which infectivity is retained, Among the many detergents examined, only treatment,vith the ionic detergent sodium dodecyl sulf ate (SDS) or Sarkosyl followed by sonication dispersed prion rods whic h are composed of PrP 27-30, an N-terminally truncated form of PrPSc, After ultracentrifugation at 100,000 X g for 1 h, similar to 30% of th e PrP 27-30 and scrapie infectivity were found in the supernatant, whi ch was fractionated by sedimentation through 5 to 20% sucrose gradient s, Near the top of the gradient, spherical particles with an observed sedimentation coefficient of similar to 6S, similar to 10 nm in diamet er and composed of four to sis PrP 27-30 molecules, were found, The sp heres could be digested with proteinase K and exhibited little, if any , scrapie infectivity. When the prion rods were disrupted in SDS and t he entire sample was fractionated by sucrose gradient centrifugation, a lipid-rich fraction at the meniscus composed of fragments of rods an d 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-helic al content; addition of 25% acetonitrile induced aggregates high in be ta sheet but remaining devoid of infectivity, Although the highly puri fied spherical oligomers of PrP 27-30 lack infectivity, they may provi de an excellent substrate for determining conditions of renaturation u nder which prion particles regain infectivity.