High-pressure NMR reveals close similarity between cold and alcohol protein denaturation in ubiquitin

Abstract

Proteins denature not only at high, but also at low temperature as well as high pressure. These denatured states are not easily accessible for experiment, because usually heat denaturation causes aggregation, whereas cold or pressure denaturation occurs at temperatures well below the freezing point of water or pressures above 5 kbar, respectively. Here we have obtained atomic details of the pressure-assisted, cold-denatured state of ubiquitin at 2,500 bar and 258 K by high-resolution NMR techniques. Under these conditions, a folded, native-like and a disordered state exist in slow exchange. Secondary chemical shifts show that the disordered state has structural propensities for a native-like N-terminal β-hairpin and α-helix and a nonnative C-terminal α-helix. These propensities are very similar to the previously described alcohol-denatured (A-)state. Similar to the A-state, (15)N relaxation data indicate that the secondary structure elements move as independent segments. The close similarity of pressure-assisted, cold-denatured, and alcohol-denatured states with native and nonnative secondary elements supports a hierarchical mechanism of folding and supports the notion that similar to alcohol, pressure and cold reduce the hydrophobic effect. Indeed, at nondenaturing concentrations of methanol, a complete transition from the native to the A-state can be achieved at ambient temperature by varying the pressure from 1 to 2,500 bar. The methanol-assisted pressure transition is completely reversible and can also be induced in protein G. This method should allow highly detailed studies of protein-folding transitions in a continuous and reversible manner.

Fig. 1.

(A) ¹H-¹⁵N HSQC spectrum of uniformly ¹⁵N/¹³C-labeled ubiquitin at 258 K and 2,500 bar (pH 6.5). Peaks are labeled with assignment information for the folded state (green) and the cold-denatured state (blue). Negative contour lines are shown in red. The amino acid sequence of ubiquitin is indicated at the top with assigned residues underlined for both states. The spectrum was acquired for 1.5 h at a sample concentration of 1 mM on an 800-MHz spectrometer. (B) Enlarged region of the box shown in A.

Fig. 2.

Secondary chemical shifts analysis (¹³C^α, ¹³C′, ¹⁵N) of the native state of ubiquitin at low temperature and high pressure (258 K, 2,500 bar) and at ambient conditions (293 K, 1 bar, pH 6.6). Secondary structure elements of the native state are drawn at the top.

Fig. 3.

Secondary chemical shift analysis (¹³C^α, ¹³C′, ¹⁵N) of the cold-denatured state (258 K, 2,500 bar), the urea-denatured state (298 K, 1 bar), and the A-state (298 K, 1 bar) of ubiquitin. Secondary structure elements of the A-state are drawn at the top. The red bars for the ¹⁵N secondary shift of the cold-denatured state (D_258K) are shifted relative to standard referencing (black bars) by −1.41 ppm to account for the uniform pressure effect on solvent-exposed amide groups (main text).

Fig. 4.

Structural switch from native state (Left) to A-state (Right). Corresponding structural elements are coded by identical colors: N-terminal β-hairpin (orange), central α-helix (magenta), and C-terminal half (green). The C-terminal half switches from a β-sheet structure in the native state to the long α-helix α′ in the A-state. The structural scheme of the A-state is adapted from Brutscher et al. (19).

Fig. 5.

T₂/T₁ ratios calculated from 600-MHz ¹⁵N relaxation data for ubiquitin. Black curve: folded-state (258 K, 2,500 bar) T₂/T₁ ratios as a function of residue number (bottom horizontal axis). Blue curve: cold-denatured state (258 K, 2,500 bar). Red curve: A-state (300 K, 1 bar) replotted from Brutscher et al. (19). The dashed green curve shows the T₂/T₁ ratio for an isotropic rotator at 600 MHz as a function of τ_c (top horizontal axis). Secondary structure elements of the A-state are drawn at the bottom.

Fig. 6.

(A) Pressure-induced unfolding of ubiquitin in 45% methanol at 308 K ¹H-¹⁵N HSQC recorded on a 600-MHz spectrometer at pressures of 1 (dark blue), 500 (blue), 1,000 (green), 1,500 (magenta), 2,000 (orange), and 2,500 bar (red), respectively. (B) Expanded region of A showing the transition in the glycine region. Resonances are labeled with assignment information corresponding to the native state (dark blue) or the pressure-induced A-state (red). (C) Secondary chemical shift analysis (¹³C^α, ¹³C′, ¹⁵N) of the pressure-induced unfolded state of ubiquitin in 45% methanol (308 K, 2,500 bar). The secondary shifts are almost identical to those of the A-state (Fig. 3). Secondary structure elements of the A-state are drawn at the top.

Fig. P1.

High-resolution NMR experiments show that the free energy landscape of ubiquitin changes under cold and pressure denaturation in a similar way as under alcohol denaturation. These conditions cause an opening of the structure and induce a structural ensemble where certain parts of native secondary structure are preserved whereas others change to nonnative forms. These individual secondary structure elements undergo independent segmental motions.