Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion

Abstract

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use (15)N, , and (13)CO NMR R(2) relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R(2) dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75-4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N&lrarr2;I1&lrarr2;MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N → I1 (36 s(-1)) and MG → I1 (26 s(-1)), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R(2) dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N → MG transition. The experiments also identify regions of energetic frustration that "crack" during unfolding and impede the refolding process.

Fig. 1.

Sensitivity of N-state ¹⁵N chemical shifts and R_ex relaxation rates to changes in pH. (A) Magnitude of ¹⁵N chemical shift changes between pH 5.9 and 4.95, color-coded to highlight His (red); residues contacting His (orange); and residues contacting the disordered F helix, FG loop, and N-terminal region of helix G (blue); other residues are colored green. The solid black rectangles at the top of the figure depict helices in apoMb based on a TALOS+ (30) analysis of , ¹⁵N, ¹³CO, , and chemical shifts (6), whereas the boundaries of the F, G, and H helices in holoMb are shown as open rectangles. (B) ¹⁵N R_ex (at a static magnetic field of 11.7 T) of deuterated apoMb at 35 °C; pH 5.9 (blue), pH 4.9 (red). R_ex was estimated from the difference in R_2eff at the lowest and highest 1/τ_cp values. The green squares on the horizontal axis indicate contact sites with helix F in holoMb, and the circles identify His residues. Yellow squares indicate the N-terminal region of helix G.

Scheme 1.

Fig. 2.

Chemical shift differences obtained from R₂ dispersion show that apoMb populates the MG state below pH 5. (A) Representative R₂ dispersion curves for apoMb at pH 4.95, 35 °C at a static magnetic field of 18.8 T: ¹⁵N-SQ (red), ¹H-SQ (purple), ¹H¹⁵N-ZQ (green), and ¹H¹⁵N-DQ (blue), showing fits to a three-state exchange model. Data acquired at 11.7 T were included in the global fit, but are omitted from the plots for clarity. Correlation of amide ¹⁵N (B) and (C) equilibrium chemical shift differences (ppm) between the N (pH 4.95) and MG (pH 4.1) states, |Δδ_N,MG|, and the chemical shift differences |Δω_N,MG| obtained from a global fit of the dispersion curves to the linear model N⇆I1⇆MG. A line of slope 1 is shown as a visual guide. Some scatter in the correlation plots for amide ¹⁵N and resonances is to be expected because of unavoidable differences in sample conditions, required to obtain spectra of the equilibrium MG (pH 4.1, 50 °C, 10% ethanol) (8).

Fig. 3.

Probing backbone conformational changes using ¹³CO R₂ dispersion. (A) Representative ¹³CO (black) and ¹⁵N (blue) R₂ dispersion curves collected on a single sample of ¹³C, ¹⁵N-apoMb, pH 4.75, at a static magnetic field of 18.8 T. Data acquired at a static field strength of 14.1 T were included in the global fit but are omitted from the figure for clarity. The solid curves represent three-state global fits to the exchange model N⇆I1⇆MG. (B) Correlation between Δω_N,MG (ppm) obtained from a global three-state fit to ¹³CO and ¹⁵N R₂ dispersion data at pH 4.75, and |Δδ_N,MG| determined from 3D NMR data under equilibrium conditions (pH 4.75 and 4.1 for N and MG, respectively). The line shows a linear fit to the data (R² = 0.88, slope 0.93). A description of parameter constraints and uncertainty analysis is in the SI Text.

Fig. 4.

Schematic free energy diagram for three-state transient unfolding (N⇆I1⇆MG) of apoMb at pH 4.95 (solid) and two-state unfolding (N⇆I1) at pH 5.5 (dashed). Barrier heights were calculated from the rates in Table 1 and Table S1 using transition-state theory and a standard assumption for the prefactor for protein folding (35). The rate-limiting barriers are marked with asterisks. The free energies of the I1 and MG states are 1.2 and 0.9 kcal/mol, respectively, above the N state.

Fig. 5.

Sites of local unfolding and structural rearrangement in the transient MG. (A) Mapping of ¹⁵N Δω_N,MG chemical shift differences onto the structure of holoMb. The protein backbone is represented as a tube. Residues with smaller than average Δω_N,MG are colored red, whereas those with above-average shifts are colored blue and vary in thickness and color saturation according to the magnitude of Δω_N,MG. (B) Secondary structure and RCI-S² parameter (32) for the transient MG state and the N state. The secondary structure of the MG state was predicted by TALOS+ (30) from , ¹⁵N, and ¹³CO chemical shifts (derived from Δω_N,MG as described in SI Text). A more complete dataset (, ¹⁵N, ¹³CO, , shifts) was available for the N state; however, the helical boundaries and RCI-S² for N were not significantly altered when predicted from a limited set of , ¹⁵N, ¹³CO shifts. The rectangles depict the location of helical structure in each state; the thickness of each rectangle is proportional to the population of helix. The hatched lines indicate the small population of transient helical structure in the C- and E-helix regions of MG. (C) Changes in secondary structure accompanying the N⇆MG transition, mapped to the structure of holoMb. Residues predicted to be helical by TALOS+ are red. The population of helix in the MG ensemble is indicated by the tube radius, with a larger radius indicating higher population. Flexible regions with RCI S² < 0.7 are blue, and coil regions with S² > 0.7 are green. A white backbone trace indicates regions for which no predictions are available. The His24 and His119 side chains are shown as pink spheres in A and C. The figure was prepared using the program MolMol (36) from the coordinates of holoMb (Protein Data Bank ID code 1MBC).