Fluorescence quenching in the DsbA protein fromEscherichia coli: Complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

Sillen, Alain; Hennecke, Jens; Roethlisberger, Daniela; Glockshuber, Rudi; Engelborghs, Yves

doi:10.1002/(sici)1097-0134(19991101)37:2<253::aid-prot10>3.0.co;2-j

Cited by 38 publications

(29 citation statements)

References 54 publications

(62 reference statements)

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“…S6G-H). RSNOs, several amino acid sidechains, and the peptide backbone can quench fluorescence 4445 . Mere mono-nitrosation therefore seems sufficient to alter the conformation of one or more buried tryptophans.…”

Section: Resultsmentioning

confidence: 99%

S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR)

et al. 2016

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The free radical nitric oxide (NO•) regulates diverse physiological processes from vasodilation in humans to gas exchange in plants. S-nitrosoglutathione (GSNO) is considered a principal nitroso reservoir due to its chemical stability. GSNO accumulation is attenuated by GSNO reductase (GSNOR), a cysteine-rich cytosolic enzyme. Regulation of protein nitrosation is not well understood since NO•-dependent events proceed without discernible changes in GSNOR expression. Because GSNORs contain evolutionarily-conserved cysteines that could serve as nitrosation sites, we examined the effects of treating plant (Arabidopsis thaliana), mammalian (human), and yeast (Saccharomyces cerevisiae) GSNORs with nitrosating agents in vitro. Enzyme activity was sensitive to nitroso donors, while the reducing agent dithiothreitol (DTT) restored activity, suggesting catalytic impairment was due to S-nitrosation. Protein nitrosation was confirmed by mass spectrometry, by which mono-, di-, and tri-nitrosation were observed, and these signals were sensitive to DTT. GSNOR mutants in specific non-zinc coordinating cysteines were less sensitive to catalytic inhibition by nitroso donors and exhibited reduced nitrosation signals by mass spectrometry. Nitrosation also coincided with decreased tryptophan fluorescence, increased thermal aggregation propensity, and increased polydispersity—properties reflected by differential solvent accessibility of amino acids important for dimerization and the shape of the substrate and coenzyme binding pockets as assessed by hydrogen-deuterium exchange mass spectrometry. Collectively, these data suggest a mechanism for NO• signal transduction in which GSNOR nitrosation and inhibition transiently permit GSNO accumulation.

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Section: Resultsmentioning

confidence: 99%

S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR)

et al. 2016

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“…Specifically, the strong tryptophan fluorescence increase of DsbA upon reduction of the catalytic disulfide bond (9, 10, 32) was retained in all variants. This is a good indication for the intactness of the tertiary structure, as fluorescence quenching by the catalytic disulfide does not occur via direct contact between a tryptophan and the disulfide but through a complex through-space energy transfer mechanism that requires the correct relative orientations and local environments of the two tryptophan residues in the protein (32,36). Overall, both the CD and fluorescence data indicate that even the biologically inactive DsbA variants 82-85 have wild type-like tertiary structures.…”

Section: Fig 2 Comparison Of the Near-uv (Top) And Far-uv (Bottom) mentioning

confidence: 89%

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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“…Experimental support for this hypothesis is strongly provided by few, but very insightful experiments [14][15][16]. In the past decade, Callis and coworkers have computationally investigated the electronic landscape associated to this electron transfer mechanism [17][18][19][20][21].…”

Section: Introductionmentioning

confidence: 99%

Fluorescence quenching of tryptophan and tryptophanyl dipeptides in solution

P.¹,

L.²

2011

JBPC

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We report measurements of fluorescence quantum yields of tryptophan, tryptophanylaspartate and tryptophanylarginine in several solvents as well as in aqueous solutions over a wide range of pH. We aim to test a computational model developed by Callis and coworkers of fluorescence quantum yield, which postulates that quenching in tryptophan arises from energy loss due to an electron transfer from the aromatic system of tryptophan to one of the amides in the protein backbone. Since the electron transfer state is expected to be high in energy, normally this would not be a possible outcome, but because of its large dipole, such a state should be more accessible in polar solvents. In addition, conditions of low (high) pH, which result in a net positive (negative) charge for the terminal amine (carboxyl) should result in an increase (decrease) of electron transfer rates and low (high) quantum yields. The observed results confirm the predictions of the model.

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Fluorescence quenching in the DsbA protein fromEscherichia coli: Complete picture of the excited-state energy pathway and evidence for the reshuffling dynamics of the microstates of tryptophan

Cited by 38 publications

References 54 publications

S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR)

S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of S-Nitrosoglutathione Reductase (GSNOR)

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

Fluorescence quenching of tryptophan and tryptophanyl dipeptides in solution

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