Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR

Abstract

The characterization of residual structures persistent in unfolded proteins in concentrated denaturant solution is currently an important issue in studies of protein folding because the residual structure present, if any, in the unfolded state may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the hydrogen/deuterium (H/D)-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride. We employed a dimethylsulfoxide (DMSO)-quenched H/D-exchange NMR technique with the use of spin desalting columns, which allowed us to perform a quick medium exchange from 6 M guanidinium chloride to a quenching DMSO solution. Based on the backbone resonance assignment of ubiquitin in the DMSO solution, we successfully investigated the H/D-exchange kinetics of 60 identified peptide amide groups in the ubiquitin sequence. Although a majority of these amide groups were not protected, certain amide groups involved in a middle helix (residues 23-34) and an N-terminal β-hairpin (residues 2-16) were significantly protected with a protection factor of 2.1-4.2, indicating that there were residual structures in unfolded ubiquitin and that these amide groups were more than 52% hydrogen bonded in the residual structures. We show that the hydrogen-bonded residual structures in the α-helix and the β-hairpin are formed even in 6 M guanidinium chloride, suggesting that these residual structures may function as a folding initiation site to guide the subsequent folding reactions of ubiquitin.

Figure 1

The ¹H–¹⁵N HSQC spectrum of uniformly ¹³C/¹⁵N-labeled ubiquitin in the DMSO solution (94.5% DMSO-d₆/0.5% DCA-d₂/5% H₂O (pH 5.4)) recorded on the ¹H 800-MHz NMR instrument at 25°C.

Figure 2

The H/D-exchange curves for Val5 (A), Asn25 (B), Gln40 (C), and Glu51 (D) in 6 M GdmCl at pH^∗ 3.3 and 15°C. The solid lines are the theoretical curves best-fitted to a single exponential function (Eq. 1). A broken line in each panel indicates the theoretically estimated peak volume after complete exchange (i.e., Y(∞) in Eq. 1), and an asterisk (^∗) in each panel, located between (1 and 2) × 10⁸ of the peak volume, indicates the experimentally observed value after heating the sample at 50°C for 30 min. Because the reaction mixtures contained 10% H₂O, the final peak volumes did not reach zero. The k_obs-values for the four residues are as follows: (A) (8.7 ± 0.8) × 10⁻³ min⁻¹, (B) (10.5 ± 1.2) × 10⁻² min⁻¹, (C) (4.6 ± 0.4) × 10⁻² min⁻¹, and (D) (1.2 ± 0.1) × 10⁻² min⁻¹.

Figure 3

For a Figure360 author presentation of Fig. 3, see the figure legend at https://doi.org/10.1016/j.bpj.2020.10.003. (A) The P-values of unfolded ubiquitin in 6 M GdmCl (pH^∗ 3.3 and 15°C) are plotted versus the residue number. The dashed line indicates the P-value of 3. The amino acid residues with P-values larger than 2 and 3 are indicated in pink and red, respectively. The locations of the secondary structures in native ubiquitin (PDB: 1UBQ) are shown by arrows (β-strands) and open rectangles (helices). (B) Mapping of the three-dimensional structure of native ubiquitin (PDB: 1UBQ) with residues showing the P-values. The red gradient indicates the scale of the P-value, which is defined by a scale bar at th bottom right corner of the panel. The proline residues and the residues that could not be used as probes because of severe broadening or overlapping are shown in black. To see this figure in color, go online.

Figure 4

(A) The H-bonding network observed in native ubiquitin (PDB: 1UBQ). Closer views of (B) the middle α-helix, (C) the N-terminal β-hairpin, (D) the one-turn 3₁₀ helix (Pro37–Gln40), and (E) the type II β-turn (Gln62–Ser65) and the one-turn 3₁₀ helix (Leu56–Tyr59) are shown. The H-bonds of the NH protons of Thr7, Val17, Lys27, Ala28, Lys29, Asp32, Lys33, Glu34, Gln40, Tyr59, and Ser65 with the CO groups of their counterparts are shown as green lines. The local H-bonds formed by the NH protons of Lys11, Asn25, and Glu51 with the side-chain atoms of Thr7, Thr22, and Tyr59, respectively, are shown as brown lines. The red gradient indicates the same scale of the P-value as shown in Fig. 3. To see this figure in color, go online.