Study of protein folding under native conditions by rapidly switching the hydrostatic pressure inside an NMR sample cell

Abstract

In general, small proteins rapidly fold on the timescale of milliseconds or less. For proteins with a substantial volume difference between the folded and unfolded states, their thermodynamic equilibrium can be altered by varying the hydrostatic pressure. Using a pressure-sensitized mutant of ubiquitin, we demonstrate that rapidly switching the pressure within an NMR sample cell enables study of the unfolded protein under native conditions and, vice versa, study of the native protein under denaturing conditions. This approach makes it possible to record 2D and 3D NMR spectra of the unfolded protein at atmospheric pressure, providing residue-specific information on the folding process. ¹⁵N and ¹³C chemical shifts measured immediately after dropping the pressure from 2.5 kbar (favoring unfolding) to 1 bar (native) are close to the random-coil chemical shifts observed for a large, disordered peptide fragment of the protein. However, ¹⁵N relaxation data show evidence for rapid exchange, on a ∼100-μs timescale, between the unfolded state and unstable, structured states that can be considered as failed folding events. The NMR data also provide direct evidence for parallel folding pathways, with approximately one-half of the protein molecules efficiently folding through an on-pathway kinetic intermediate, whereas the other half fold in a single step. At protein concentrations above ∼300 μM, oligomeric off-pathway intermediates compete with folding of the native state.

Keywords: NMR spectroscopy; folding intermediate; high pressure; protein folding; ubiquitin.

Fig. 1.

Schematic representation of the pressure jump NMR apparatus. The protein solution is contained in a zirconia NMR sample tube, rated for 3 kbar of pressure, and is connected through stainless-steel tubing and a valve to a 125-mL reservoir containing pressurized hydraulic fluid (mineral oil or mineral spirits). A second valve connects the tubing to a vessel that is kept at 1 bar under an atmosphere of N₂ gas. Both air-activated valves are operated under spectrometer control. A hydraulic pump is used to recycle decompressed fluid back into the high-pressure reservoir. The entire assembly is mounted on a pneumatic lift for entry of the sample into the NMR magnet.

Fig. 2.

Observation of protein folding and unfolding by NMR spectroscopy. (A) Schematic diagram of a time course experiment for the measurement of N successive low-pressure 2D HSQC spectra (and M high-pressure spectra) to follow the protein folding (unfolding) process on a residue-by-residue basis. Hydrostatic pressure is marked in green. At time points τ_a + nT after each pressure switch, N (or M) FIDs are recorded, and collection of the entire series of FIDs is repeated L times, with L being the number of t₁ increments times the number of scans (typically 2) used for signal averaging. Final 2D NMR spectra then correspond to the state of the protein at times τ_a + nT after the pressure switch. (B) Subset of the series of spectra recorded after the pressure was dropped to 1 bar to monitor the appearance of folded protein signals (blue) and the disappearance of the unfolded state resonances (red), with colors added manually. Residues are labeled by their one-letter residue code and number, with U and F denoting the unfolded and native state, respectively. (C) Time dependence of the resonance intensities of well-resolved cross-peaks. (D and E) Data analogous to B and C but monitoring the unfolding of the protein after pressure is jumped from 1 bar to 2.5 kbar. Spectra were recorded at 5 °C (set temperature at high pressure) for a sample containing 0.3 mM ubiquitin in 25 mM phosphate buffer, pH 6.4. The time dependence of signal appearance and disappearance at other temperatures and pressures is included in SI Appendix, Fig. S1.

Fig. 3.

Observation of protein folding by zz-exchange spectroscopy. (A) Schematic timing diagram of the pulse sequence. At the end of a high-pressure equilibration period (12 s), the ¹H magnetization is transferred to ¹⁵N_z magnetization by a refocused INEPT pulse scheme. Conversion to transverse ¹⁵N magnetization and subsequent t₁ evolution, followed by a pulse that stores a cos(ω_Nt₁) fraction of this magnetization back to z, is initiated at time τ_d after the pressure drop, thereby encoding the ¹⁵N frequencies present at τ_d. At a fixed time, T, after the pressure drop, this encoded ¹⁵N magnetization is transferred back to the amide proton for ¹H detection. T is chosen sufficiently long (330 ms) that most of the protein has folded at the time of detection. (B–F) Small regions of the HSQC spectra, recorded on a 280 μM ²H/¹⁵N/¹³C-enriched ubiquitin sample at indicated τ_d delay durations after the pressure drop. All spectra correlate the frequency of the detected amide proton in the folded protein to the ¹⁵N frequency of either the unfolded (red) or folded (blue) protein, with resonance intensities proportional to the fractions populating the unfolded and native states. Note that colors have been added manually. Residues are labeled by their one-letter residue code and number, with U and F denoting the unfolded and native state, respectively. (G) Time dependence of the resonance intensities observed in B–F. Fitted time constants show faster disappearance of resonances of the unfolded state than appearance of the folded final spectrum.

Fig. 4.

Temperature and pressure dependence of ubiquitin folding and unfolding. (A) Disappearance of intensity of unfolded and appearance of folded protein signals as a function of time after the pressure is dropped from 2.5 kbar to 1 bar. Signal intensities are obtained with the scheme shown in Fig. 2 for a sample containing 0.3 mM ubiquitin, pH 6.4, at 5 °C. Intensities are averaged over a set of well-resolved resonances. Data for 15 and 25 °C are shown in SI Appendix, Fig. S1. (B) Arrhenius activation energies of ubiquitin folding, derived from the data shown in A and SI Appendix, Fig. S1. (C) Appearance of unfolded state and disappearance of folded state signals at 15 °C, 2.5 kbar. Data at 5 and 25 °C are shown in SI Appendix, Fig. S1. (D) Arrhenius activation energies of ubiquitin unfolding at 2.5 kbar, derived from the data shown in C. (E and F) Pressure dependence of ubiquitin unfolding at 15 °C. (E) Disappearance of folded protein signals and appearance of unfolded protein signals after jumping from atmospheric pressure to 2.0 kbar. Data measured at other pressures (1.8, 2.2, and 2.5 kbar) are shown in SI Appendix, Fig. S1C. (F) Plot of the protein unfolding rate against pressure, to derive the activation volume ΔV*_U. Unfolding rates are obtained from the fits shown in SI Appendix, Fig. S1C. Scatter in the graph is dominated by uncertainty in the coarsely regulated pressure.

Fig. 5.

Folding of ubiquitin as monitored by the C^δ2Η₃ resonance of L50. (A) Concentration dependence of folding kinetics as monitored by the intensity, I(t), of the native L50 C^δ2H₃ intensity at 15 °C. Solid lines are best fits to I(t) = I(0) + F (1 − exp(t/T_m)) + (1 − F − I(0)) (1 − exp(t/T_o)), where F is the fraction folding with the fast time constant of the monomeric protein, T_m = 370 ms, and T_o is the time constant (3.54 s) of the slowly recovering fraction. I(0) is the protein fraction that does not unfold at 2.5 kbar (∼2.5%). (B) Partitioning of the monomeric (blue) and oligomeric (gray) fractions at various total protein concentrations. (C) Series of ¹H NMR spectra (150 μM, 25 °C), at indicated times after the pressure is dropped from 2.5 kbar to 1 bar. (D) Upfield region of a pressure jump 2D NOESY spectrum (100-ms mixing time), where the pressure drop occurs 40 ms before the start of ¹H t₁ evolution. The strong cross-peak between the F₁ frequency of the L50^I C^δ2H₃ (at −0.2 ppm) and the native L50^F C^δ2H₃ resonance (at −0.3 ppm) shows that the folding intermediate is on-pathway. (E) Intensity of the native L50 C^δ2H₃ resonance (blue) and the folding intermediate (green) as a function of refolding time. The intensity has been converted to concentration, using the intensity of the fully folded protein as a reference. Solid lines are the best-fit solutions calculated using the indicated rate constants.

Fig. 6.

Measurement of chemical shifts of unfolded ubiquitin at 1 bar, by 3D NMR. Data are recorded for a 300 μM sample of U(¹⁵N,¹³C,²H) ubiquitin in 25 mM phosphate buffer, pH 6.4, 22 °C. (A) Schematic diagram of the pressure jump NMR experiment. The preparation period (Prep.) consists of a long delay (8 s) at high pressure, terminated by an efficient transfer of ¹H^N to z magnetization of either ¹⁵N, or the ¹³Cʹ or ¹³C^α of the preceding residue. For measurement of unfolded ¹H^N frequencies at 1 bar, the ¹H^N t₁ evolution period starts immediately after the pressure drop, and precedes transfer to ¹⁵N. Immediately after the pressure drop, ¹H^N, ¹⁵N, ¹³Cʹ, or ¹³C^α evolution (t₁) modulates this z magnetization by cos(ω_Xt₁) or sin(ω_Xt₁), where X = ¹H^N, ¹⁵N, ¹³Cʹ, or ¹³C^α, followed by transfer of X_z to in-phase ¹⁵N_z magnetization. At time T = 270 ms after the pressure drop, ¹⁵N magnetization (following a t₂ evolution period) is transferred to ¹H^N for detection. Note that ¹H detection starts always at the same time point, T, after the initial pressure drop. (B) Projection of the 3D [¹⁵N_unfolded, ¹⁵N_folded, ¹H_folded] spectrum on the ¹⁵N_unfolded–¹⁵N_folded plane, correlating the ¹⁵N chemical shifts in the unfolded and folded states. (C) Experimental secondary chemical shifts in parts per million for the unfolded protein at 1 bar (orange bars, Δδ_unfolded). For comparison; the secondary chemical shifts of the folded protein, scaled down by a factor of 5, are also shown (blue points, Δδ_folded). The secondary chemical shifts are calculated relative to values of the pressure-denatured state at 2.5 kbar, adjusted to 1 bar using the random-coil pressure dependence values of Kalbitzer and coworkers (56, 57). (D) Correlation plots for residues 2–18 between the unfolded 1-bar secondary chemical shifts (y axis) and folded 1-bar secondary chemical shifts (x axis). The Pearson correlation coefficient, R, and the slope S of a linear regression analysis are marked in each panel.

Fig. 7.

Measurement of ¹⁵N transverse relaxation in the unfolded state at 1 bar. (A) Schematic timing diagram of the pulse sequence. The preparation period (Prep.) consists of a high-pressure equilibration period (10 s) followed by transfer of ¹H magnetization to ¹⁵N_z by a refocused INEPT pulse scheme. The pressure is then dropped to 1 bar for a fixed duration T (T = 125 ms). Immediately following the pressure drop, an adiabatic scheme is used to bring ¹⁵N magnetization parallel to the spin-lock field for a variable duration τ_SL, before conversion back to the z axis. A pair of ¹H 180° pulses, applied at τ_SL/4 and 3τ_SL/4, is used for removal of cross-correlated relaxation. When τ_SL is increased, the time for storage of ¹⁵N along the z axis decreases, and the measured decay rate corresponds to R_1ρ – R₁. After the pressure is jumped back to 2.5 kbar, the amplitude of the ¹⁵N_z magnetization is recorded as a regular 2D HSQC spectrum in the unfolded state. Note that this spectrum also contains resonances for the folded state, resulting from protein that folded during the low-pressure period, and has not yet unfolded at the time of ¹H detection. These signals are discarded. (B) Representative decay curves for selected residues, measured using a ¹⁵N spin-lock field of 2 kHz for a 150 μM ¹⁵N-labeled ubiquitin in standard buffer, with the sample temperature set to 15 °C, but the actual temperature being ∼3 °C lower during the brief low-pressure period where the relaxation is actually measured. (C) ¹⁵N relaxation rates for the unfolded protein at 1 bar. R_1ρ rates shown have been derived from the fitted rates by removing the R₁ and offset contributions, as described in the text. Rates measured at three different spin-lock RF field strengths are all faster than seen at high pressure (aqua; SI Appendix, Fig. S4), indicative of conformational exchange contributions, R_ex, that scale with the strength of the spin-lock field (Inset). R_ex is calculated as $R_{\mathrm{ex}}={R_{1\rho }}_{\mathrm{RF}}^{1\mathrm{bar}\hspace{0.17em}}–{R_{1\rho }}_{2\hspace{0.17em}\mathrm{kHz}}^{2.5\hspace{0.17em}\mathrm{kbar}}$, where ${R_{1\rho }}_{\mathrm{RF}}^{1\mathrm{bar}}$ is the rate measured in the pressure jump experiments at the indicated RF field strength, and ${R_{1\rho }}_{2\hspace{0.17em}\mathrm{kHz}}^{2.5\hspace{0.17em}\mathrm{kbar}}$ is the R_1ρ rate measured in the pressure-denatured state (2.5 kbar) using a 2-kHz spin-lock field.