The characterization of residual structures persistent in unfolded proteins is an important issue in studies of protein folding, because the residual structures present, if any, may form a folding initiation site and guide the subsequent folding reactions. Here, we studied the residual structures of the isolated B domain (BDPA) of staphylococcal protein A in 6 M guanidinium chloride. BDPA is a small three-helix-bundle protein, and until recently its folding/unfolding reaction has been treated as a simple two-state process between the native and the fully unfolded states. We employed a dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange 2D NMR techniques with the use of spin desalting columns, which allowed us to investigate the H/D-exchange behavior of individually identified peptide amide (NH) protons. We obtained H/D-exchange protection factors of the 21 NH protons that form an ?-helical hydrogen bond in the native structure, and the majority of these NH protons were significantly protected with a protection factor of 2.0–5.2 in 6 M guanidinium chloride, strongly suggesting that these weakly protected NH protons form much stronger hydrogen bonds under native folding conditions. The results can be used to deduce the structure of an early folding intermediate, when such an intermediate is shown by other methods. Among three native helical regions, the third helix in the C-terminal side was highly protected and stabilized by side-chain salt bridges, probably acting as the folding initiation site of BDPA. The present results are discussed in relation to previous experimental and computational findings on the folding mechanisms of BDPA.
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[NMR paper] Protein structure prediction using residue-resolved protection factors from hydrogen-deuterium exchange NMR
Protein structure prediction using residue-resolved protection factors from hydrogen-deuterium exchange NMR
Hydrogen-deuterium exchange (HDX) measured by nuclear magnetic resonance (NMR) provides structural information for proteins relating to solvent accessibility and flexibility. While this structural information is beneficial, the data cannot be used exclusively to elucidate structures. However, the structural information provided by the HDX-NMR data can be supplemented by computational methods. In previous work, we developed an algorithm in Rosetta to predict structures using...
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[NMR paper] Protein Structure Prediction from NMR Hydrogen-Deuterium Exchange Data
Protein Structure Prediction from NMR Hydrogen-Deuterium Exchange Data
Amide hydrogen-deuterium exchange (HDX) has long been used to determine regional flexibility and binding sites in proteins; however, the data are too sparse for full structural characterization. Experiments that measure HDX rates, such as HDX-NMR, have far higher throughput compared to structure determination via X-ray crystallography, cryo-EM, or a full suite of NMR experiments. Data from HDX-NMR experiments encode information on the protein structure, making HDX a prime candidate to be...
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[NMR paper] Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR.
Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR.
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Biophys J. 2020 Oct 14;:
Authors: Yagi-Utsumi M, Chandak MS, Yanaka S, Hiranyakorn M, Nakamura T, Kato K, Kuwajima K
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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...
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[NMR paper] Measurement and Characterization of Hydrogen-Deuterium Exchange Chemistry Using Relaxation Dispersion NMR Spectroscopy.
Measurement and Characterization of Hydrogen-Deuterium Exchange Chemistry Using Relaxation Dispersion NMR Spectroscopy.
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J Phys Chem B. 2018 03 01;122(8):2368-2378
Authors: Khirich G, Holliday MJ, Lin JC, Nandy A
Abstract
One-dimensional heteronuclear relaxation dispersion NMR...
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[NMR paper] Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.
Lactose Binding Induces Opposing Dynamics Changes in Human Galectins Revealed by NMR-Based Hydrogen-Deuterium Exchange.
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Molecules. 2017 Aug 16;22(8):
Authors: Chien CH, Ho MR, Lin CH, Hsu SD
Abstract
Galectins are ?-galactoside-binding proteins implicated in a myriad of biological functions. Despite their highly conserved carbohydrate binding motifs with essentially identical structures, their...
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[NMR paper] [Interactions between proteins and cation exchange adsorbents analyzed by NMR and hydrogen/deuterium exchange technique].
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Sheng Wu Gong Cheng Xue Bao. 2014 Sep;30(9):1454-63
Authors: Wang K, Hao D, Qi S, Ma G
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In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H-NMR shows that protein unfolding accelerated...
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Quantification of protein backbone hydrogen-deuterium exchange rates by solid state N
Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy
Abstract We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that backbone amides in the microcrystalline α-spectrin SH3 domain exchange rather slowly with the solvent (with exchange rates negligible within the individual 15Nâ??T 1 timescales). We observed chemical exchange for 6...
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Quantification of protein backbone hydrogen-deuterium exchange rates by solid state N
Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy.
Related Articles Quantification of protein backbone hydrogen-deuterium exchange rates by solid state NMR spectroscopy.
J Biomol NMR. 2010 Oct 20;
Authors: Del Amo JM, Fink U, Reif B
We present the quantification of backbone amide hydrogen-deuterium exchange rates (HDX) for immobilized proteins. The experiments make use of the deuterium isotope effect on the amide nitrogen chemical shift, as well as on proton dilution by deuteration. We find that...
The B Domain of Protein A Retains Residual Structures in 6 M Guanidinium Chloride as Revealed by Hydrogen/Deuterium-Exchange NMR Spectroscopy
Linear Mode
