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 bhairpin (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 a-helix and the b-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.

DMSO-Quenched H/D-Exchange 2D NMR Spectroscopy and Its Applications in Protein Science

“…HNCAHA experiments using 13 C/ 15 N-double-labeled proteins. We applied the improved DMSO-quenched 2D NMR method to investigate the H/D-exchange behavior of the E. coli co-chaperonin GroES at pH* 6.5 (or 7.5) and 25 °C [135] and unfolded ubiquitin at pH* 3.2 and 15.0 °C in 6 M GdmCl [136], In the following, we will describe the study on the H/Dexchange behavior of unfolded ubiquitin as a case study.…”

“…The presence of the residual structure, if any, in the unfolded state thus invalidates the Levinthal paradox, because such residual structure may form a folding initiation site and guide the subsequent folding reactions. We therefore studied the H/D-exchange behavior of unfolded human ubiquitin in 6 M GdmCl by the DMSO-quenched H/D-exchange 2D NMR method with the use of spin desalting columns [136]. Although the persistence of residual structures in unfolded proteins in concentrated denaturant has been reported for several different proteins [50,[147][148][149][150][151][152][153], the present method enabled us to estimate the p values of individually identified NH protons, including nonprotected NH protons in the N state [136].…”

“…These results may be compared with the present results of the H/D-exchange behavior of unfolded ubiquitin in 6 M GdmCl. To analyze the H/D-exchange kinetics of individually identified NH protons of ubiquitin, we first made their spectral assignments in the DMSO solution [136]. Using a combination of 3D spectral measurements, we successfully assigned all the peaks observed in the HSQC spectrum.…”

“…We then investigated the H/D-exchange kinetics of all the individual NH groups. Excluding NH groups whose residues could not be used as probes due to severe broadening or overlapping, we successfully followed the H/D-exchange kinetics of Molecules 2022, 27, 3748 7 of 17 60 NH protons [136]. These 60 NH protons include not only the protons stably protected in the native structure but also nonprotected NH protons.…”

“…We used 2D 1 H- 15 N HSQC spectra for the NMR analysis, and hence the sample protein was 15 N-labeled, and the assignment of the HSQC cross peaks was carried out by three-dimensional (3D) HN(CA)NNH, HNCA, HN(CO)CA, HNCO, CBCA(CO)NH, and HNCAHA experiments using 13 C/ 15 N-double-labeled proteins. We applied the improved DMSO-quenched 2D NMR method to investigate the H/D-exchange behavior of the E. coli co-chaperonin GroES at pH* 6.5 (or 7.5) and 25 3.2 and 15.0 • C in 6 M GdmCl [136], In the following, we will describe the study on the H/D-exchange behavior of unfolded ubiquitin as a case study.…”