Early intermediate in human prion protein folding as evidenced by ultrarapid mixing experiments

Abstract

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.

Scheme I

Figure 1. The time-course of tryptophan fluorescence changes during the refolding of huPrP90-231 in 50 mM phosphate buffer, pH 7.0, in the presence of 1.1 M urea

The refolding kinetics was measured by continuous-flow mixing at 5°C and a protein concentration of 40 μM. Solid lines represent the best fit of the kinetic data to a single exponential (A) and double exponential function (B). The residuals of the fits are shown above kinetic traces. The equilibrium fluorescence value for the native state was used as a constraint for both the single and double exponential fits.

Figure 2. Representative continuous-flow kinetic traces for huPrP90-231 refolding in varying concentrations of urea at pH 7 (50 mM phosphate buffer)

Solid lines represent the best fit of kinetic traces to a double exponential function. Numbers at each curve indicate final concentrations of urea.

Figure 3. Urea concentration dependence of rate constants for folding/unfolding of huPrP90-231 at pH 7

(■) and (●) represent rate constants for fast and slow refolding phases, respectively, as observed in continuous flow experiments; (○) represent rate constants for the single folding/unfolding phase observed in stopped-flow experiments. All rate constants were measured at 5°C in 50 mM phosphate buffer, pH 7. Lines represent best fit according to a three-state folding model (Scheme I).

Figure 4. Urea concentration dependence of rate constants for folding/unfolding of the diseases-associated F198S mutant of huPrP90-231 at pH 7

(■) and (●) represent rate constants for fast and slow refolding phases, respectively, as observed in continuous flow experiments; (○) represent rate constant for the single folding/unfolding phase observed in stopped-flow experiments. All rate constants were measured at 5°C in 50 mM phosphate buffer, pH 7. Lines represent best fit according to a three-state folding model (Scheme I).

Figure 5. Time-dependent evolution of the population of the intermediate state, I, during refolding of wild-type huPrP90-231 (left panel) and the F198S variant (right panel) at different urea concentrations in 50 mM phosphate buffer, pH 7

Simulations were performed as described in the Materials and Methods section. Numbers by each curve indicate the concentration of urea.

Figure 6. Urea concentration dependence of rate constants for folding/unfolding of huPrP90-231 at pH 4.8

(■) and (●) represent rate constants for fast and slow refolding phases, respectively, as observed in continuous flow experiments; (○) represent rate constant for the single folding/unfolding phase observed in stopped-flow experiments. All rate constants were measured at 5°C in 50 mM sodium acetate buffer, pH 4.8. Lines represent best fit according to a three-state folding model (Scheme 1).