NR AWDK
AU Colby,D.W.; Wille,H.; Legname,G.; Prusiner,S.B.
TI Conformational engineering of synthetic prions
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions S-07
PT Konferenz-Poster
AB The advent of synthetic prions, which are formed from recombinant prion protein (recPrP) folded into ß-rich conformations, is enabling new insights into the structural basis of prion infectivity (Legname, et al., Science, 2004). Recent evidence suggests that the conformational stability of prions isolated from infected mice correlates directly with the incubation period for the onset of CNS dysfunction (Legname, et al., manuscript in preparation). We sought to establish whether it is possible to generate misfolded PrP conformers with varying conformational stabilities in vitro, and if so, whether these conformers result in different incubation periods upon intracerebral injection into mice. recPrP of mouse residues 89-231 was incubated in solution, and the formation of PrPsc-like conformers was monitored by Thioflavin T fluorescence. In hopes of generating distinct conformations, we varied the composition of the solution with respect to denaturant and salt concentration, pH, and the presence of protein-stabilizing additives; the temperature of incubation was also varied. We have identified conditions for the formation of recPrP conformers that are less stable, or less resistant to guanidine and temperature denaturation, as assessed by loss of Thioflavin T fluorescence and by the fraction of soluble recPrP. Differences in fiber morphology were observed by electron microscopy. Whether these differences will correspond to changes in the incubation period remains to be determined. The ability to create distinct conformations of PrP in vitro and to correlate their structural features to prion disease pathology will be of enormous value to understanding the structural basis of prion infectivity.
AD Institute for Neurodegenerative Diseases, University of California, San Francisco, USA. E-mail: dcolby@ind.ucsf.edu
SP englisch
PO Italien