A simple way to measure protein refolding rates in water

Daniel E. Otzen; Mikael Oliveberg

doi:10.1006/jmbi.2001.5039

A simple way to measure protein refolding rates in water

^*Corresponding author for this work

Interdisciplinary Nanoscience Center - INANO-MBG, iNANO-huset

Research output: Contribution to journal/Conference contribution in journal/Contribution to newspaper › Journal article › Research › peer-review

37 Citations (Scopus)

Abstract

Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with α-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.

Original language	English
Journal	Journal of Molecular Biology
Volume	313
Issue	3
Pages (from-to)	479-483
Number of pages	5
ISSN	0022-2836
DOIs	https://doi.org/10.1006/jmbi.2001.5039
Publication status	Published - 26 Oct 2001

Keywords

Chevron plots
Cyclodextrin
Protein folding
SDS
Stopped-flow

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title = "A simple way to measure protein refolding rates in water",

abstract = "Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with α-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.",

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AU - Otzen, Daniel E.

AU - Oliveberg, Mikael

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N2 - Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with α-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.

AB - Refolding of proteins is traditionally carried out either by diluting the denaturant-unfolded protein into buffer (GdmCl-jump) or by mixing the acid-denatured protein with strong buffer (pH-jump). The first method does not allow direct measurement of folding rates in water since the GdmCl cannot be infinitely diluted, and the second method suffers from the limitation that many proteins cannot be pH-denatured. Further, some proteins do not refold reversibly from low pH where they get trapped as aggregation prone intermediates. Here, we present an alternative approach for direct measurement of refolding rates in water, which does not rely on extrapolation. The protein is denatured in SDS, and is then mixed with α-cyclodextrin, which rapidly strips SDS molecules from the protein, leaving the naked unfolded protein to refold.

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KW - Stopped-flow

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A simple way to measure protein refolding rates in water

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Keywords

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