Reduction-potential of Glutathione

J, Rost; Rapoport, S.

doi:10.1038/201185a0

Cited by 167 publications

(107 citation statements)

References 7 publications

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“…An additional advantage of the Cys-Cys system is that it permits a comparably more active consolidation, which is catalysed by trace amounts of mixed disulphides and free cysteine that exist in the state of equilibrium. These differences can be explained by the relative redox potential (E.') of the GSH/GSSG (-0.24 V) and Cys/Cys-Cys (-0.22 V) systems (Rost and Rapoport, 1964;Jocelyn, 1967). A thermodynamically favoured GSSG is less potent at promoting the disulphide formation of proteins.…”

Section: Resultsmentioning

confidence: 99%

Controlling the speed of hirudin folding

Chang

1994

Biochemical Journal

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The folding of hirudin undergoes an initial stage of non-specific packing, followed by consolidation (re-organization) of partially packed intermediates to attain the native structure [Chatrenet and Chang (1993) J. Biol. Chem. 268, 20988-20996]. Non-specific packing leads to the formation of scrambled hirudins as folding intermediates. A systematic study was carried out to search for conditions which would selectively control and enhance the processes of packing and consolidation. It is demonstrated here that under optimized conditions, including the use of cystine/cysteine and protein disulphide isomerase, the folding of hirudin in vitro can be achieved quantitatively within 30 s.

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Section: Resultsmentioning

confidence: 99%

Controlling the speed of hirudin folding

Chang

1994

Biochemical Journal

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“…These results imply that roGFP is neither sufficiently sensitive to visualize the redox dynamics at the physiological level in the cytosol nor sufficiently sensitive to distinguish the differences in the redox state between normal VOL. 30,2010 REDOX STATE AND PEROXISOME DEFICIENCY 3765…”

Section: Discussionmentioning

confidence: 99%

“…2A). Based on the Nernst equation with an E 0 value of Ϫ240 mV for the GSH/GSSG redox couple (30), the redox midpoint potential of the C probe was determined to be Ϫ213 mV at pH 7.0 and 30°C, which was close to the value of the mammalian cytosolic redox potential (10). From the titration curve, the redox potential within the range of ϳϪ180 to Ϫ250 mV can be estimated from the FRET ratio ( Fig.…”

Section: Development and Characterization Of Redoxfluor As A Redox Inmentioning

confidence: 99%

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Yano

Oku

Akeyama

et al. 2010

Molecular and Cellular Biology

102

101

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Reactive oxygen species are generated within peroxisomes during peroxisomal metabolism. However, due to technological difficulties, the intraperoxisomal redox state remain elusive, and the effect of peroxisome deficiency on the intracellular redox state is controversial. A newly developed, genetically encoded fluorescence resonance energy transfer (FRET) probe, Redoxfluor, senses the physiological redox state via its internal disulfide bonds, resulting in a change in the conformation of the protein leading to a FRET response. We made use of Redoxfluor to measure the redox states at the subcellular level in yeast and Chinese hamster ovary (CHO) cells. In wild-type peroxisomes harboring an intact fatty acid ␤-oxidation system, the redox state within the peroxisomes was more reductive than that in the cytosol, despite the fact that reactive oxygen species were generated within the peroxisomes. Interestingly, we observed that the redox state of the cytosol of cell mutants for peroxisome assembly, regarded as models for a neurological metabolic disorder, was more reductive than that of the wild-type cells in yeast and CHO cells. Furthermore, Redoxfluor was utilized to develop an efficient system for the screening of drugs that moderate the abnormal cytosolic redox state in the mutant CHO cell lines for peroxisome assembly without affecting the redox state of normal cells.

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“…Random Mutagenesis and Screening-The plasmid pDsbA3-CC30/ 33AA for periplasmic production of the inactive DsbA variant Cys 30 3 Ala-Cys 33 3 Ala was derived after amplification of the mutant gene from a previously described plasmid (26) and replacement of the dsbA wild type gene in pDsbA3 (27) via the restriction sites NdeI and BamHI. Plasmids pDsbA3 and pDsbA3-CC30/33AA thus both contain the trc promoter and lac operator sequence for inducible gene expression and an f1 replication origin.…”

Section: Methodsmentioning

confidence: 99%

“…In addition, His 32 presumably contributes about one pK a unit to the lowered pK a of Cys 30 , possibly by formation of a hydrogen bond between the Cys 30 thiolate and N␦1 (7, 16 -18). In addition, possible hydrogen bonds with the thiol of Cys 33 , the amide hydrogen of His 32 , and the amide hydrogen of Cys 33 further stabilize the Cys 30 thiolate (7).…”

mentioning

confidence: 99%

Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Philipps¹,

Glockshuber²

2002

Journal of Biological Chemistry

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DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E o ‫؍‬ ؊122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys 30 -Pro 31 -His 32 -Cys 33 ) is primarily due to an extremely low pK a value (3.4) of Cys 30 , which is stabilized by the partial positive dipole charge of the active-site helix ␣1 (residues 30 -37). We have randomized all non-cysteine residues of helix ␣1 (residues 31, 32, and 34 -37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines ␣-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below ؊210 mV. All inactive variants had redox potentials lower than ؊210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.

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Reduction-potential of Glutathione

Cited by 167 publications

References 7 publications

Controlling the speed of hirudin folding

Controlling the speed of hirudin folding

A Novel Fluorescent Sensor Protein for Visualization of Redox States in the Cytoplasm and in Peroxisomes

Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

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