title: Relationship between conformational stability and amplification efficiency of prions.
abstract: Recent studies demonstrated that the efficiency, rate, and yield of prion amplification in vitro could be substantially improved by supplementing protein misfolding cyclic amplification (PMCA) with Teflon beads [Gonzalez-Montalban et al. (2011) PLoS Pathog. 7, e1001277]. Here we employed the new PMCA format with beads (PMCAb) to gain insight into the mechanism of prion amplification. Using a panel of six hamster prion strains, the effect of beads on amplification was found to be strain-specific, with the largest improvements in efficiency observed for strains with the highest conformational stability. This result suggests a link between PrP(Sc) conformational stability and its fragmentation rate and that beads improved amplification by assisting fragmentation. Furthermore, while exploring the PrP(Sc)-independent bead effect mechanism, a synergy between the effects of RNA and beads on amplification was observed. Consistent with previous studies, amplification of all six hamster strains tested here was found to be RNA-dependent. Under sonication conditions used for PMCA, large RNA molecules were found to degrade into smaller fragments of a size that was previously shown to be the most effective in facilitating prion conversion. We speculate that sonication-induced changes in RNA size distribution could be one of the rate-limiting steps in prion amplification.
authors.affiliation: Center for Biomedical Engineering and Technology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Nuria
authors.initials: N
authors.lastname: Gonzalez-Montalban
authors.forename: Natallia
authors.initials: N
authors.lastname: Makarava
authors.forename: Regina
authors.initials: R
authors.lastname: Savtchenko
authors.forename: Ilia V
authors.initials: IV
authors.lastname: Baskakov
mesh_terms: D000818:Animals; D006224:Cricetinae; D008647:Mesocricetus; D051379:Mice; D008810:Mice, Inbred C57BL; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D018620:PrPSc Proteins / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability
chemical_list: D018620:PrPSc Proteins