Cytochrome c folding pathway: kinetic native-state hydrogen exchange

Abstract

Native-state hydrogen exchange experiments under EX1 conditions can distinguish partially unfolded intermediates by their formation rates and identify the amide hydrogens exposed and protected in each. Results obtained define a cytochrome c intermediate seen only poorly before and place it early on the major unfolding pathway. Four distinct unfolding steps are found to be kinetically ordered in the same pathway sequence inferred before.

Fig 1.

Unfolding by native-state HX. (A) Cyt c with color-coded unfolding units and its suggested folding/unfolding pathway. (B) Equilibrium vs. kinetic NHX. Equilibrium NHX, done under EX2 conditions, can distinguish intermediates (I but not I′) by their equilibrium ΔG and by their m^o, ΔH^o, or ΔV^o in denaturant, temperature, or pressure-dependent HX experiments, respectively. The kinetic NHX method, done under EX1 conditions, can distinguish intermediates (I or I′) by their unfolding rates and their sensitivity to the high pH necessarily used. Unfolding barriers may be detected independently of whether the barrier is seen (rate-limiting) in the usual folding experiment or whether the intermediate formed is stable relative to U, unlike eNHX. Both methods identify the amino acids protected and exposed in each PUF by their HX labeling.

Fig 2.

Simulated HX results. (A and B) At low pH where exchange is in the EX2 limit, HX rate increases 10-fold for each pH unit. At high pH, HX rate limits at k_op (EX1 limit). (C) Simulation of expected curves when the HX exposure time is kept constant and pH is varied (k_op = 10 s⁻¹, k_cl = 10⁴ s⁻¹, k_int = 10⁸ s⁻¹). The arrow shows the EX2 to EX1 transition point (k_ch = k_cl). An experimental curve in the gray region most sensitively measures the opening/closing parameters. At high pH, a different EX1 pattern can be produced when k_op increases with pH (black line in Fig. 5).

Fig 3.

High pH denaturation of Cyt c (no urea). (A) Above pH 12, denaturation occurs in two steps with a populated intermediate. A partial unfolding removes 45% of the CD₂₂₂ of the native protein and recovers all of the fluorescence of the U state (corrected for Trp pH titration and quenching). Both probes show the same midpoint (pK 12.8) and slope (1.5). Equilibrium unfolding to U occurs above pH 13. (B) The kinetics of intermediate formation. The black curve in Fig. 5A is constructed from Fl data in 1.3 M urea. The zero urea data are shown here because CD₂₂₂ is obscured in high pH urea.

Fig 4.

Kinetic NHX as a function of time. HX behavior is shown for marker residues in the Blue and Green units (A) and the Yellow unit (B), and for all of the protected residues in the Red unit (C). Conditions were pH 10.14 (open symbols, dashed lines) and pH 11.24 (closed symbols, bold lines) in 0.5 M KCl at 20°C with 1.3 M urea to slow reclosing.

Fig 5.

Kinetic NHX as a function of pH, and protein unfolding. (A) Measured amide hydrogens in the four unfolding units of Cyt c (1.3 M urea; data without urea are very similar), with constant pulse labeling time of 75 ms. Dotted lines show hydrogens previously found to exchange by way of local fluctuations; solid lines show previously identified marker hydrogens. The reference black curve shows the HX labeling that would be determined by the formation of the high pH intermediate, assuming EX1 exchange. Data for each hydrogen are fit by Eqs. 7, 1, and 2 plus the high pH intermediate formation. Black points for the Yellow unit are from Fig. 4B. Residues 70 and 91 at the two ends of the Red segment (71–85; 91 H-bonds to 87) begin to show plateauing, like the Red unit. His-33 is protected in the U state (1). (B) The individual residues in the Red unit without and with added urea. All exchange as markers, with the subglobal Red loop unfolding, because of the high pH destabilizing condition. Different hydrogens plateau earlier or later depending on their k_int value. Labeling rate increases sharply at pH 12 and above because of formation of the high pH intermediate. Black points (Lower) are from the time-dependent HX data in Fig. 4C. (C) Rollover of the Cyt c unfolding chevron compared with HX unfolding rates for the Red unit. The unfolding arm at pH 7 and the rollover at pH 7 and 10 are shown. (At lower denaturant, unfolding at pH 10 becomes biphasic as in Fig. 3; data shown are monophasic.) Back extrapolation of the pH 10 rollover rate is shown at ±1σ. To convert 1.3 M urea to equivalent GdmCl, a factor of 2.5 was used (41); the stronger GdmCl denaturant was necessary to access the rollover region. (D) Fractional H-labeling at the given pH (75 ms). Bars represent the average H-label of the marker residues for each unfolding unit, taken from A. The sequential decrease in opening rates is best explained by a sequential unfolding pathway, as in E. (E) Classical stepwise folding/unfolding pathway for Cyt c. The diagram illustrates barrier heights and well depths that will generate the Red, Yellow, and Green unfoldings indicated by the kNHX results. [In this case the relationship τ_obs = Στ_i closely holds, where the rate of first passage opening (τ) is measured by EX1 HX and the summation is over all prior openings.]