A Native-like Intermediate Serves as a Branching Point between the Folding and Aggregation Pathways of the Mouse Prion Protein

Abstract

Transient folding intermediates and/or partially unfolded equilibrium states are thought to play a key role in the formation of protein aggregates. However, there is only indirect evidence linking accumulation of folding intermediates to aggregation, and the underlying mechanism remains to be elucidated. Here, we show that a partially unfolded state of the prion protein accumulates both as a stable equilibrium state at acidic pH (A-state) and as a late folding intermediate. With a time resolution of approximately 60 μs, we systematically studied the kinetics of folding and unfolding, starting from various initial conditions including the U-, N-, and A-states. Quantitative modeling showed that the observed kinetic data are completely consistent with a sequential four-state mechanism where the A-state is a late folding intermediate. Combined with previous evidence linking A-state accumulation to aggregation, the results indicate that this native-like state serves as a branching point between the folding and aggregation pathways.

Figure 1. Structural and thermodynamic characteristics of the A-state

Far-UV CD (A), near- UV CD (B) and Trp-fluorescence (C) spectra for the N-state at pH 4.5 (red), the A-state at pH 2.0 (blue), and the U-state at pH 2.0 with 3 M urea (dotted black), respectively. (D) Thermal unfolding of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue) monitored via the change in mean residue ellipcity at 222 nm. The solid lines represent least-squared fits to van’t Hoff’s equation, where the equilibrium constant between two states is described by the exponential of (ΔH_wf – ΔS·T)/RT. [ΔH_wf (kcal/mol) and ΔS (cal/mol·K)] of N-and A-states are (42 ± 4 and 122 ± 11) and (16 ± 1 and 48 ± 2), respectively. (E) ¹³Cα-chemical shift indexes of the N-state at pH 4.5 (red) and the A-state at pH 2.0 (blue). (F) ¹³Cα-chemical shift indexes of the A-state plotted onto the 3D-structure of mouse PrP(121–231) (PDB:1AG2). The unassigned residues are shown in gray.

Figure 2. Folding and unfolding kinetics

(A) Representative kinetic traces for folding of mouse PrP at pH 4.5 in the presence of 0.5–2.5 M urea, starting from conditions favoring the U-state (pH 2.0, 3.0 M urea). (B) Representative traces for unfolding at pH 4.5 in the presence of 5.0–7.0 M urea, starting from conditions favoring the N-state (pH 4.8, 2.0 M urea). The solid lines represent least-squared fits to a single exponential function. (C) Chevron plots in the absence (filled circles, ●) or presence (empty squares, ◻) of 0.17 M Na₂SO₄. The solid and dotted lines represent curves generated by assuming a three-state scheme (Table S1).

Figure 3. Kinetics of folding and unfolding of the A-state

(A) Representative folding/unfolding kinetic traces at pH 4.5 in the presence of 0–7.0 M urea starting from conditions favoring the Astate (pH 2.0). The solid lines represent least-squared fits to a double exponential function: f (t) / f (A)= A_f exp(–λ_ft)+ A_s exp(–λ_st)+ f_∞ (Eq. 1) (B) Chevron plot. The filled circles (●) and the filled triangles (▲) represent λ_f and λ_s in Eq. 1, respectively. For comparison, the apparent rate constants in Figure 2C are shown as empty squares (◻). (C) Amplitude plot. Circles (●), down-triangles (▼), and up-triangles (▲) represent f_∞, (f_∞+A_s), and (f_∞+A_s+A_f) from Eq. 1, respectively. The solid curves in Figure 3B & C represent curves reproduced by assuming the U↔I↔A↔N scheme (Table 1). The fluorescence intensity of the U, I, A, and N-states at 0 M urea is 1.01, 1.30, 1.05, and 1.05 respectively. The slope against urea concentration (M⁻¹) of the U, I, A, and N-states is 0.23, 0.10, 0.05, and 0.05, respectively. (Inset) Equilibrium urea titration curve obtained under the same conditions. The dotted line represents the baseline N-state. (D) Free-energy diagram. The activation free energies of the transitions, Ea=RT ln(k/A₀), were calculated from k_IA and k_AN (Table 1) values using a pre-exponential factor A₀ = 10⁶ (s^-1).

Figure 4. Folding and unfolding kinetics predicted by the U↔I↔A↔N scheme

The folding kinetics at 0 M urea (A) and the unfolding kinetics at 6 M urea (B) were calculated by solving the four-state mechanism using the parameters in Table 1 and the legend of Figure 3. The upper and lower panels represent the time-dependent fluorescence change and the fraction of each state, respectively.

Figure 5. A schematic free-energy landscape for folding and oligomerization of PrP

In this scheme, unfolded PrP^C (U) folds into the N structure via two intermediate states: the I- and A-states (black line). Oligomerization and aggregation starts from a late folding intermediate, the A-state (red line). The A-state is a key intermediate from which the folding and oligomerization pathways diverge. F, Q₁, and Q₂ represent the Gibbs free energy, the first reaction coordinate, and the second reaction coordinate, respectively.