Crystal structure of the DsbA protein required for disulphide bond formation in vivo

Nature ◽  
1993 ◽  
Vol 365 (6445) ◽  
pp. 464-468 ◽  
Author(s):  
Jennifer L. Martin ◽  
James C. A. Bardwell ◽  
John Kuriyan
Keyword(s):  
Crystal Structure ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Disulphide Bond Formation

Crystallization of DsbA, an Escherichia coli Protein Required for Disulphide Bond Formation in Vivo

1993 ◽  
Vol 230 (3) ◽  
pp. 1097-1100 ◽  
Author(s):  
Jennifer L. Martin ◽  
Gabriel Waksman ◽  
James C.A. Bardwell ◽  
Jon Beckwith ◽  
John Kuriyan
Keyword(s):  
Escherichia Coli ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Disulphide Bond Formation

scholarly journals TriPer, an optical probe tuned to the endoplasmic reticulum tracks H2O2 consumption by glutathione

2016 ◽  
Author(s):  
Eduardo Pinho Melo ◽  
Carlos Lopes ◽  
Peter Gollwitzer ◽  
Stephan Lortz ◽  
Sigurd Lenzen ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Biology ◽  
Genetic Manipulation ◽  
Bond Formation ◽  
Ectopic Expression ◽  
Optical Probe ◽  
Disulphide Bond ◽  
In Vivo Studies ◽  
Disulphide Bond Formation

AbstractThe fate of H2O2 in the endoplasmic reticulum (ER) has been inferred indirectly from the activity of ER localized thiol oxidases and peroxiredoxins, in vitro, and the consequences of their genetic manipulation, in vivo. Here we report on the development of TriPer, a vital optical probe sensitive to changes in the concentration of H2O2 in the thiol-oxidizing environment of the ER. Consistent with the hypothesized contribution of oxidative protein folding to H2O2 production, ER-localized TriPer detected an increase in the luminal H2O2 signal upon induction of pro-insulin (a disulfide bonded protein of pancreatic β-cells), which was attenuated by the ectopic expression of catalase in the ER lumen. Interfering with glutathione production in the cytosol by buthionine sulfoximine (BSO) or enhancing its localized destruction by expression of the glutathione-degrading enzyme ChaC1 in lumen of the ER, enhanced further the luminal H2O2 signal and eroded β-cell viability. Tracking ER H2O2 in live cells points to an unanticipated role for glutathione in H2O2 turnover.Significance statementThe presence of millimolar glutathione in the lumen of the endoplasmic reticulum has been difficult to understand purely in terms of modulation of protein-based disulphide bond formation in secreted proteins. Over the years hints have suggested that glutathione might have a role in reducing the heavy burden of hydrogen peroxide (H2O2) produced by the luminal enzymatic machinery for disulphide bond formation. However, limitations in existing in vivo H2O2 probes have rendered them all but useless in the thiol-oxidizing ER, precluding experimental follow-up of glutathione’s role ER H2O2 metabolism.Here we report on the development and mechanistic characterization of an optical probe, TriPer that circumvents the limitations of previous sensors by retaining specific responsiveness to H2O2 in thiol-oxidizing environments. Application of this tool to the ER of an insulin-producing pancreatic b-cells model system revealed that ER glutathione antagonizes locally-produced H2O2 resulting from the oxidative folding of pro-insulin.This study presents an interdisciplinary effort intersecting cell biology and chemistry: An original redox chemistry concept leading to development of a biological tool, broadly applicable for in vivo studies of H2O2 metabolism in the ER. More broadly, the concept developed here sets a precedent for applying a tri-cysteine relay system to discrimination between various oxidative reactants, in complex redox milieux.

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

1995 ◽  
Vol 17 (6) ◽  
pp. 1035-1044 ◽  
Author(s):  
Kim R. Hardie ◽  
Angela Schulze ◽  
Michael W. Parker ◽  
J. Thomas Buckley
Keyword(s):  
Bond Formation ◽  
Disulphide Bond ◽  
Disulphide Bond Formation ◽  
Vibrio Spp

scholarly journals Influence of the peptide-chain length on disulphide-bond formation in neurohypophysial hormones and analogues

1978 ◽  
Vol 173 (2) ◽  
pp. 403-409 ◽  
Author(s):  
G Moore
Keyword(s):  
Acetic Acid ◽  
Arginine Vasopressin ◽  
Solid Phase ◽  
Bond Formation ◽  
Gel Filtration ◽  
Arginine Vasotocin ◽  
Disulphide Bond ◽  
Peptide Chain ◽  
Phase Method ◽  
Disulphide Bond Formation

(8-Arginine)vasopressin, (8-arginine)vasotocin, oxytocin and oxypressin, the ‘ring’ derivatives pressinamide and tocinamide, and the extended-chain analogues Pro-Arg-Val-(8-arginine)vasopressin and (8-arginine)vasopressinoyl-Ala-Met-Ala-NH(2), were synthesized by the solid-phase method and purified by sequential gel filtration on Sephadex G-15 in 50% acetic acid and 0.2M-acetic acid. Controlled oxidation of the thiol groups of the reduced peptides obtained after deprotection with sodium in liquid ammonia gave rise to products that depended on the length of the peptide chain: (i) nonapeptides gave monomer and dimer species, (ii) hexapeptides produced mixtures containing higher polymers, and (iii) dodecapeptides gave predominantly monomer with some dimerized material. The evidence suggests that the presence of the acyclic tail tripeptide in the nonapeptide hormones induces a conformation in the preceding hexapeptide that favours the formation of an intramolecular disulphide bond. For (8-arginine)vasopressin, intramolecular disulphide-bond formation is enhanced by extension of the peptide chain from either the N- or the C-terminus. The possible significance of these studies to neurohypophysial hormone-prohormone relationships is discussed.

scholarly journals Chemical modification of Escherichia coli succinyl-CoA synthetase with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine

1983 ◽  
Vol 215 (3) ◽  
pp. 513-518 ◽  
Author(s):  
A R S Prasad ◽  
J Ybarra ◽  
J S Nishimura
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Adenine Nucleotide ◽  
Bond Formation ◽  
Disulphide Bond ◽  
Thiol Groups ◽  
Binding Region ◽  
Nucleotide Analogue ◽  
Disulphide Bond Formation ◽  
Succinyl Coa Synthetase

Escherichia coli succinyl-CoA synthetase (EC 6.2.1.5) was irreversibly inactivated on incubation with the adenine nucleotide analogue 5′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). Optimal inactivation by 5′-FSBA took place in 40% (v/v) dimethylformamide. ATP and ADP protected the enzyme against inactivation by 5′-FSBA, whereas desulpho-CoA, an analogue of CoA, did not. Inactivation of succinyl-CoA synthetase by 5′-FSBA resulted in total loss of almost four thiol groups per alpha beta-dimer, of which two groups appeared to be essential for catalytic activity. 5′-FSBA at the first instance appeared to interact non-specifically with non-essential thiol groups, followed by a more specific reaction with essential thiol groups in the ATP(ADP)-binding region. Plots of the data according to the method of Tsou [(1962) Sci. Sin. 11, 1535-1558] revealed that, of the two slower-reacting thiol groups, only one was essential for catalytic activity. When succinyl-CoA synthetase that had been totally inactivated by 5′-FSBA was unfolded in acidic urea and then refolded in the presence of 100 mM-dithiothreitol, 85% of the activity, in comparison with the appropriate control, was restored. These data are interpreted to indicate that inactivation of succinyl-CoA synthetase by 5′-FSBA involves the formation of a disulphide bond between two cysteine residues. Disulphide bond formation likely proceeds via a thiosulphonate intermediate between 5′-p-sulphonylbenzoyladenosine and one of the reactive thiol groups of the enzyme.

Mechanisms and catalysts of disulphide bond formation in proteins

1995 ◽  
Vol 13 (1) ◽  
pp. 18-23 ◽  
Author(s):  
Thomas E. Creighton ◽  
André Zapun ◽  
Nigel J. Darby
Keyword(s):  
Bond Formation ◽  
Disulphide Bond ◽  
Disulphide Bond Formation
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