Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments.
An important step toward understanding the mechanism of the PrP(C)-to-PrP(Sc) conversion is to elucidate the folding pathway(s) of the prion protein. On the basis of stopped-flow measurements, we recently proposed that the prion protein folds via a transient intermediate formed on the submillisecond time scale, and mutations linked to familial diseases result in a pronounced increase in the population of this intermediate. Here, we have extended these studies to continuous-flow measurements using a capillary mixing system with a time resolution of approximately 100 micros. This allowed us to directly observe two distinct phases in folding of the recombinant human prion protein 90-231, providing unambiguous evidence for rapid accumulation of an early intermediate (with a time constant of approximately 50 micros), followed by a rate-limiting folding step (with a time constant of approximately 700 micros). The present study also clearly demonstrates that the population of the intermediate is significantly increased at mildly acidic pH and in the presence of urea. A similar three-state folding behavior was observed for the Gerstmann-Straussler-Scheinker disease-associated F198S mutant, in which case the population of an intermediate was greatly increased as compared to that of the wild-type protein. Overall, the present data strongly suggest that this partially structured intermediate may be a direct monomeric precursor of the misfolded PrP(Sc) oligomer.
Apetri
Adrian C
AC
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106, USA.
Maki
Kosuke
K
Roder
Heinrich
H
Surewicz
Witold K
WK
D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011328:Prions / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D013816:Thermodynamics; D013997:Time Factors; D014508:Urea / Q000737:chemistry
D010446:Peptide Fragments; D011328:Prions; C514520:prion protein (91-231), human; D014508:Urea