Document Detail


The use of spin desalting columns in DMSO-quenched H/D-exchange NMR experiments.
MedLine Citation:
PMID:  23339068     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Dimethylsulfoxide (DMSO)-quenched hydrogen/deuterium (H/D)-exchange is a powerful method to characterize the H/D-exchange behaviors of proteins and protein assemblies, and it is potentially useful for investigating non-protected fast-exchanging amide protons in the unfolded state. However, the method has not been used for studies on fully unfolded proteins in a concentrated denaturant or protein solutions at high salt concentrations. In all of the current DMSO-quenched H/D-exchange studies of proteins so far reported, lyophilization was used to remove D2 O from the protein solution, and the lyophilized protein was dissolved in the DMSO solution to quench the H/D exchange reactions and to measure the amide proton signals by two-dimensional nuclear magnetic resonance (2D NMR) spectra. The denaturants or salts remaining after lyophilization thus prevent the measurement of good NMR spectra. In this article, we report that the use of spin desalting columns is a very effective alternative to lyophilization for the medium exchange from the D2 O buffer to the DMSO solution. We show that the medium exchange by a spin desalting column takes only about 10 min in contrast to an overnight length of time required for lyophilization, and that the use of spin desalting columns has made it possible to monitor the H/D-exchange behavior of a fully unfolded protein in a concentrated denaturant. We report the results of unfolded ubiquitin in 6.0M guanidinium chloride.
Authors:
Mahesh S Chandak; Takashi Nakamura; Toshio Takenaka; Tapan K Chaudhuri; Maho Yagi-Utsumi; Jin Chen; Koichi Kato; Kunihiro Kuwajima
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2013-02-11
Journal Detail:
Title:  Protein science : a publication of the Protein Society     Volume:  22     ISSN:  1469-896X     ISO Abbreviation:  Protein Sci.     Publication Date:  2013 Apr 
Date Detail:
Created Date:  2013-03-20     Completed Date:  2013-08-26     Revised Date:  2014-04-01    
Medline Journal Info:
Nlm Unique ID:  9211750     Medline TA:  Protein Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  486-91     Citation Subset:  IM    
Copyright Information:
Copyright © 2013 The Protein Society.
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MeSH Terms
Descriptor/Qualifier:
Chemical Fractionation / instrumentation*,  methods*
Deuterium Exchange Measurement / methods*
Dimethyl Sulfoxide / chemistry*
Freeze Drying
Humans
Models, Chemical
Nuclear Magnetic Resonance, Biomolecular / methods*
Salts / isolation & purification*
Ubiquitin / analysis,  chemistry
Chemical
Reg. No./Substance:
0/Salts; 0/Ubiquitin; YOW8V9698H/Dimethyl Sulfoxide
Comments/Corrections

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