| The use of spin desalting columns in DMSO-quenched H/D-exchange NMR experiments. | |
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MedLine Citation:
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PMID: 23339068 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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DMSO-quenched 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 D(2) 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 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 D(2) 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 6.0 M guanidinium chloride. |
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Authors:
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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:
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Type: JOURNAL ARTICLE Date: 2013-1-22 |
Journal Detail:
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Title: Protein science : a publication of the Protein Society Volume: - ISSN: 1469-896X ISO Abbreviation: Protein Sci. Publication Date: 2013 Jan |
Date Detail:
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Created Date: 2013-1-22 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9211750 Medline TA: Protein Sci Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2013 The Protein Society. |
Affiliation:
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Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan; Department of Functional Molecular Science, School of Physical Sciences, Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan. |
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