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

Abstract

Hydrogen/deuterium (H/D) exchange combined with two-dimensional (2D) NMR spectroscopy has been widely used for studying the structure, stability, and dynamics of proteins. When we apply the H/D-exchange method to investigate non-native states of proteins such as equilibrium and kinetic folding intermediates, H/D-exchange quenching techniques are indispensable, because the exchange reaction is usually too fast to follow by 2D NMR. In this article, we will describe the dimethylsulfoxide (DMSO)-quenched H/D-exchange method and its applications in protein science. In this method, the H/D-exchange buffer is replaced by an aprotic DMSO solution, which quenches the exchange reaction. We have improved the DMSO-quenched method by using spin desalting columns, which are used for medium exchange from the H/D-exchange buffer to the DMSO solution. This improvement has allowed us to monitor the H/D exchange of proteins at a high concentration of salts or denaturants. We describe methodological details of the improved DMSO-quenched method and present a case study using the improved method on the H/D-exchange behavior of unfolded human ubiquitin in 6 M guanidinium chloride.

The B domain of protein A retains residual structures in 6 M guanidinium chloride as revealed by hydrogen/deuterium‐exchange NMR spectroscopy

“…We used the DMSO‐quenched method in the present H/D‐exchange experiments, in which the H/D‐exchange reaction of 15 N‐labeled BDPA was quenched by the DMSO/D 2 O solution (94.5% (v/v) DMSO‐ d 6 /5.0% D 2 O/0.5%(v/v) dichloroacetic acid‐ d 2 (DCA‐ d 2 ), pH* 5.4) and the NMR spectra of the protein were measured in the DMSO/D 2 O solution (Chandak et al 2013 ; Kuwajima et al 2022 ). To analyze the H/D‐exchange kinetics of individually identified NH groups of 15 N‐labeled BDPA, we first made their spectral assignment in the 1 H– 15 N HSQC spectrum of the protein in the DMSO/H 2 O solution (94.5% (v/v) DMSO‐ d 6 /5.0% (v/v) H 2 O/0.5% (v/v) DCA‐ d 2 , pH 5.4).…”

“…DMSO‐quenched H/D‐exchange 2D NMR spectroscopy with the use of spin desalting columns (Chandak et al 2013 ; Kuwajima et al 2022 ) is thus very useful for detecting residual secondary structures in the U state and deducing a possible structured folding intermediate for proteins, especially when the proteins fold very rapidly with a time constant much less than ~1 ms, so that the intermediate cannot be detected by conventionally used techniques such as stopped‐flow or pulse‐labeling H/D‐exchange 2D NMR.…”

“…The use of spin desalting columns for the medium exchange in the DMSO‐quenched H/D‐exchange experiments is superior to conventionally used refolding and lyophilization. The details of the use of spin desalting columns were reported previously (Chandak et al 2013 ; Kuwajima et al 2022 ; Yagi‐Utsumi et al 2020 ).…”

“…These equilibrium unfolding parameters show reasonable agreement with the corresponding parameter values in previous studies (Table 1) (Myers and Oas 2001;Arora et al 2004). (Chandak et al 2013;Kuwajima et al 2022). To analyze the H/D-exchange kinetics of individually identified NH groups of 15 N-labeled BDPA, we first made their spectral assignment in the 1 H-15 N HSQC spectrum of the protein in the DMSO/H 2 O solution (94.5% (v/v) DMSO-d 6 /5.0% (v/v) H 2 O/0.5% (v/v) DCA-d 2 , pH 5.4).…”

“…Recently, we developed a new dimethylsulfoxide (DMSO)‐quenched hydrogen/deuterium (H/D)‐exchange two‐dimensional (2D)‐NMR method to detect and characterize the residual structures of a protein retained in the U state in concentrated denaturant (Chandak et al 2013 ; Kuwajima et al 2022 ). In this method, spin desalting columns are used to quickly exchange the medium (e.g., 6 M GdmCl) for a DMSO solution, which quenches the H/D‐exchange reactions (Zhang et al 1995 ), and then the 2D NMR spectra of the protein are measured in the DMSO solution.…”