Oxidative Protein Folding Is Driven by the Electron Transport System

Bader, Martin W.; Muse, Wilson B.; Ballou, David P.; Gassner, Christian; Bardwell, James C.A.

doi:10.1016/s0092-8674(00)81016-8

Cited by 366 publications

(348 citation statements)

References 37 publications

Supporting

Mentioning

340

Contrasting

Unclassified

Order By: Relevance

“…DsbA concentration was varied between 0 and 40 M, and the reactions were followed by the decrease in DsbA fluorescence at 330 nm. A membrane preparation from an E. coli strain overproducing DsbB was used to catalyze the reaction as described previously (4,35).…”

Section: Figmentioning

confidence: 99%

See 1 more Smart Citation

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

Philipps¹,

Glockshuber²

2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

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.

show abstract

Section: Figmentioning

confidence: 99%

“…DsbA-mediated disulfide bond formation in the periplasm involves oxidation of reduced, newly translocated substrate polypeptides by DsbA, followed by reoxidation of DsbA by the inner membrane protein DsbB. DsbB is in turn reoxidized by molecular oxygen through ubiquinone and terminal cytochrome oxidases (3,4).…”

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

View full text Add to dashboard Cite

show abstract

“…Disulfides are well known to play an important structural role, contributing to the maintenance of the proper folding in proteins. More recently it was found that the formation of disulfides in proteins also exerts critical regulatory functions: vital mechanisms such as protein import, regulation of signal transduction cascades, regulation of the activity of transcription factors and proper function of the mitochondrial electron transport system rely on disulfide oxidation or reduction [1][2][3][4]. Moreover, the formation of disulfides represents an early, reversible response to oxidative *Corresponding author: mastroberardinopg@upmc.edu.…”

Section: Introductionmentioning

confidence: 99%

A FRET-based method to study protein thiol oxidation in histological preparations

Mastroberardino

Orr

et al. 2008

Free Radical Biology and Medicine

View full text Add to dashboard Cite

Cysteine residues in proteins have important biological roles. For example, disulfides bonds are important structural elements; additionally, reversible oxidation of thiols to disulfides functions as a molecular switch and constitutes an early response to oxidative damage Because organs are heterogeneous structures composed of diverse cell types, there is a compelling need for a histological approach to investigate thiol oxidation in situ in order to address the role of specific cell types in oxidative imbalance. Here we describe a fluorescence technique -which can be used in association with standard immunological staining procedures -to detect variations in disulfides in histological preparations. Moreover, by monitoring the fluorescence resonance energy transfer (FRET) between a labeled specific primary antibody and the thiol probe described here, this method can detect thiol oxidation in candidate proteins of interest. When applied to an animal model of Parkinson's disease, our technique demonstrated that thiol oxidation occurs selectively in the dopaminergic neurons of the substantia nigra, the same neurons that are lost selectively in the disease. In summary, this technique provides a new, powerful tool to provide further understanding of oxidative imbalance, a phenomenon common to many diseases. Keywords fluorescence microscopy; FRET; oxidative stress; cysteine; thiols; disulfides; Parkinson's disease Thiols are the functional groups in the side chain of the amino acid, cysteine. Depending upon the oxido-reductive (redox) status of the surrounding environment, thiols exist either in the reduced form of free thiols (-SH) or oxidized to disulfides (-S-S-). Disulfides are well known to play an important structural role, contributing to the maintenance of the proper folding in proteins. More recently it was found that the formation of disulfides in proteins also exerts critical regulatory functions: vital mechanisms such as protein import, regulation of signal transduction cascades, regulation of the activity of transcription factors and proper function of the mitochondrial electron transport system rely on disulfide oxidation or reduction [1][2][3][4]. Moreover, the formation of disulfides represents an early, reversible response to oxidative *Corresponding author: mastroberardinopg@upmc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Increasing interest in the study of thiols has led to the development of a variety of technical approaches to detect thiols and disulfides. In particular, several spectrophotometry-based protocols were developed to measure t...

show abstract

“…Clearly, under the anaerobic and reducing conditions typical for the growth of strict anaerobes, a different pathway for the disposal of electrons must be imagined. Bader et al (1999) have recently shown that when a strain of E. coli with null mutations in cytochrome bd and bo oxidases was grown under anaerobic conditions in a GasPak Anaerobic System (albeit with no added reducing agent) to avoid interference from trace quantities of oxygen and with glucose as the primary carbon source, the periplasmic indicator protein β-lactamase was maintained in an oxidized form. This and related data documented that in E. coli growing under anaerobic conditions, the DsbADsbB system shuttles electrons via menaquinone to terminal acceptors such as fumarate.…”

Section: Redox State Of Dnase a In Vivomentioning

confidence: 99%

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo

MacLellan

Forsberg

2001

Microbiology

View full text Add to dashboard Cite

DNase A is a non-specific endonuclease of Fibrobacter succinogenes. The enzyme was purified to homogeneity and its properties studied both in vitro and in vivo. Magnesium but not calcium was essential for nucleolytic activity. Manganese ions substituted for magnesium but were less stimulatory. DNase A activity was markedly inhibited by either NaCl or KCl at concentrations greater than 75 mM. The enzyme had a temperature optimum of 25 SC and a pH optimum of about 7 0. Values for K m and K cat were determined to be 61 µM and 330 s N1 respectively, with a catalytic efficiency approximately threefold greater than bovine pancreatic DNase I, but 10-fold less than the Serratia marcescens NucA. DNase A was localized to the periplasm and probably exists as a monomeric species. The enzyme possessed one or more disulfide bonds. In the reduced form it had an apparent mass of 33 kDa, while in the oxidized form it was 29 kDa as estimated by SDS-PAGE. Reduction of the disulfide bonds by dithiothreitol with or without subsequent alkylation by iodoacetamide strongly inactivated the enzyme. DNase A accumulated in vivo had an apparent mass of 29 kDa, indicating that it was in an oxidized form. This is the first indication in a strict anaerobe of a functional periplasmic disulfide bond forming system, phenotypically similar to Dsb systems in facultative and aerobic bacteria.

show abstract

Oxidative Protein Folding Is Driven by the Electron Transport System

Cited by 366 publications

References 37 publications

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

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

A FRET-based method to study protein thiol oxidation in histological preparations

Properties of the major non-specific endonuclease from the strict anaerobe Fibrobacter succinogenes and evidence for disulfide bond formation in vivo

Contact Info

Product

Resources

About