Reductive unfolding and oxidative refolding of a Bowman–Birk inhibitor from horsegram seeds (Dolichos biflorus): evidence for ‘hyperreactive’ disulfide bonds and rate-limiting nature of disulfide isomerization in folding

2002 ◽  
Vol 1597 (2) ◽  
pp. 280-291 ◽  
Author(s):  
R.Rajesh Singh ◽  
A.G. Appu Rao
Keyword(s):  
Disulfide Bonds ◽  
Dolichos Biflorus ◽  
Oxidative Refolding ◽  
Disulfide Isomerization ◽  
Rate Limiting

scholarly journals Genetic Analysis of Disulfide Isomerization in Escherichia coli: Expression of DsbC Is Modulated by RNase E-Dependent mRNA Processing

2004 ◽  
Vol 186 (3) ◽  
pp. 654-660 ◽  
Author(s):  
Xiaoming Zhan ◽  
Junjun Gao ◽  
Chaitanya Jain ◽  
Michael J. Cieslewicz ◽  
James R. Swartz ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Disulfide Bonds ◽  
Critical Role ◽  
Chemical Mutagenesis ◽  
Selection Strategy ◽  
Rnase E ◽  
Disulfide Isomerase ◽  
Isomerase Activity ◽  
Disulfide Isomerization ◽  
Disulfide Isomerase Activity

ABSTRACT We designed a selection strategy for the isolation of Escherichia coli mutants exhibiting enhanced protein disulfide isomerase activity. The folding of a variant of tissue plasminogen activator (v-tPA), a protein containing nine disulfide bonds, in the bacterial periplasm is completely dependent on the level of disulfide isomerase activity of the cell. Mutations that increase this activity mediate the formation of catalytically active v-tPA, which in turn cleaves a p-aminobenzoic acid (PABA)-peptide adduct to release free PABA and thus allows the growth of an auxotrophic strain. Following chemical mutagenesis, a total of eight E. coli mutants exhibiting significantly higher disulfide isomerization activity, not only with v-tPA but also with two other unrelated protein substrates, were isolated. This phenotype resulted from significantly increased expression of the bacterial disulfide isomerase DsbC. In seven of the eight mutants, the upregulation of DsbC was found to be related to defects in RNA processing by RNase E, the rne gene product. Specifically, the genetic lesions in five mutants were shown to be allelic to rne, while an additional two mutants exhibited impaired RNase E activity due to lesions in other loci. The importance of mRNA stability on the expression of DsbC is underscored by the short half-life of the dsbC transcript, which was found to be only 0.8 min at 37°C in wild-type cells but was two- to threefold longer in some of the stronger mutants. These results (i) confirm the central role of DsbC in disulfide bond isomerization in the bacterial periplasm and (ii) suggest a critical role for RNase E in regulating DsbC expression.

Rapid intracellular assembly of tenascin hexabrachions suggests a novel cotranslational process

1995 ◽  
Vol 108 (4) ◽  
pp. 1761-1769 ◽  
Author(s):  
S.D. Redick ◽  
J.E. Schwarzbauer
Keyword(s):  
Disulfide Bonds ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Human Glioma ◽  
Human Glioma Cells ◽  
Amino Terminal ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting ◽  
Hexameric Form

Tenascin, an extracellular matrix protein that modulates cell adhesion, exists as a unique six-armed structure called a hexabrachion. The human hexabrachion is composed of six identical 320 kDa subunits and the structure is stabilized by inter-subunit disulfide bonds between amino-terminal segments. We have examined the biosynthesis of tenascin and its assembly into hexabrachions using pulsechase labeling of U-138 MG human glioma cells. Newly synthesized tenascin hexamers are secreted within 60 minutes of translation initiation. Intracellularly, as early as full length tenascin can be detected in pulse-labeled cell lysates, it is already in hexameric form. No precursors, such as monomers, dimers, or trimers, were identified that could be chased into hexamers. This lack of assembly intermediates suggests that nascent tenascin polypeptides associate prior to completion of translation. In contrast, fibronectin monomers in the same lysates are gradually formed into disulfide-bonded dimers. Although hexamer assembly is rapid, the rate-limiting step in secretion appears to be transport to the medial Golgi as endoglycosidase H-resistance was not detected until after a 30 minute chase. These results provide evidence for a novel co-translational mechanism of tenascin assembly which would be facilitated by its length and by the amino-terminal location of the assembly domain.

scholarly journals Division of labor among oxidoreductases: TMX1 preferentially acts on transmembrane polypeptides

2015 ◽  
Vol 26 (19) ◽  
pp. 3390-3400 ◽  
Author(s):  
Giorgia Brambilla Pisoni ◽  
Lloyd W. Ruddock ◽  
Neil Bulleid ◽  
Maurizio Molinari
Keyword(s):  
Disulfide Bonds ◽  
Protein Disulfide Isomerase ◽  
Client Protein ◽  
Redox Catalyst ◽  
The Family ◽  
Associated Proteins ◽  
Intermolecular Disulfide Bonds ◽  
Protein Disulfide ◽  
Rate Limiting ◽  
Er Membrane

The endoplasmic reticulum (ER) is the site of maturation for secretory and membrane proteins in eukaryotic cells. The lumen of the mammalian ER contains >20 members of the protein disulfide isomerase (PDI) superfamily, which ensure formation of the correct set of intramolecular and intermolecular disulfide bonds as crucial, rate-limiting reactions of the protein folding process. Components of the PDI superfamily may also facilitate dislocation of misfolded polypeptides across the ER membrane for ER-associated degradation (ERAD). The reasons for the high redundancy of PDI family members and the substrate features required for preferential engagement of one or the other are poorly understood. Here we show that TMX1, one of the few transmembrane members of the family, forms functional complexes with the ER lectin calnexin and preferentially intervenes during maturation of cysteine-containing, membrane-associated proteins while ignoring the same cysteine-containing ectodomains if not anchored at the ER membrane. As such, TMX1 is the first example of a topology-specific client protein redox catalyst in living cells.

scholarly journals BIREFRINGENCE OF SPERMATOZOA

1972 ◽  
Vol 55 (2) ◽  
pp. 489-500 ◽  
Author(s):  
James Bearden ◽  
Irwin J. Bendet
Keyword(s):  
Ethylene Glycol ◽  
Disulfide Bonds ◽  
Human Sperm ◽  
Molecular Structures ◽  
Rate Limiting Step ◽  
Ionic Bonds ◽  
Entropy Of Activation ◽  
Enthalpy And Entropy ◽  
Sperm Nuclei ◽  
Rate Limiting

In experiments designed to determine the thermal stability and bonding strength of a natural nucleoprotein structure, the loss of birefringence as a function of time and temperature was investigated for both mammalian and nonmammalian sperm nuclei. At a constant temperature, this reaction was found to be first order for both types over a range of temperatures. The methods of chemical kinetics applied to results of these reactions, called birefringence melting reactions, produced values for the enthalpy and entropy of activation in the reactions, which gave some indication of the strength of binding in the nucleoprotein structure; and these results, plus those on the influence of chemicals on the structure, were consistent with the molecular structures which have been proposed by others for the nucleoprotein complex of sperm nuclei. For both bull and human sperm in ethylene glycol, the rate-limiting step in the melting reactions appeared to be the breakage of disulfide bonds. For squid sperm in ethylene glycol, and bull or squid sperm in ethylene glycol plus ß-mercaptoethanol, the identity of this step was more ambiguous, but a possibility consistent with these and other results would be a cooperative breakage of ionic bonds.

scholarly journals Applications of catalyzed cytoplasmic disulfide bond formation

2019 ◽  
Vol 47 (5) ◽  
pp. 1223-1231 ◽  
Author(s):  
Mirva J. Saaranen ◽  
Lloyd W. Ruddock
Keyword(s):  
Disulfide Bond ◽  
Disulfide Bonds ◽  
Molecular Mechanisms ◽  
Bond Formation ◽  
Disulfide Bond Formation ◽  
Post Translational Modification ◽  
Natural Systems ◽  
Cell Factories ◽  
Engineered Systems ◽  
Disulfide Isomerization

Abstract Disulfide bond formation is an essential post-translational modification required for many proteins to attain their native, functional structure. The formation of disulfide bonds, otherwise known as oxidative protein folding, occurs in the endoplasmic reticulum and mitochondrial inter-membrane space in eukaryotes and the periplasm of prokaryotes. While there are differences in the molecular mechanisms of oxidative folding in different compartments, it can essentially be broken down into two steps, disulfide formation and disulfide isomerization. For both steps, catalysts exist in all compartments where native disulfide bond formation occurs. Due to the importance of disulfide bonds for a plethora of proteins, considerable effort has been made to generate cell factories which can make them more efficiently and cheaper. Recently synthetic biology has been used to transfer catalysts of native disulfide bond formation into the cytoplasm of prokaryotes such as Escherichia coli. While these engineered systems cannot yet rival natural systems in the range and complexity of disulfide-bonded proteins that can be made, a growing range of proteins have been made successfully and yields of homogenously folded eukaryotic proteins exceeding g/l yields have been obtained. This review will briefly give an overview of such systems, the uses reported to date and areas of future potential development, including combining with engineered systems for cytoplasmic glycosylation.

Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Biochemistry ◽  
1987 ◽  
Vol 26 (11) ◽  
pp. 3129-3134 ◽  
Author(s):  
Takashi Tsuji ◽  
Ryusuke Nakagawa ◽  
Nobuyuki Sugimoto ◽  
Kenichi Fukuhara
Keyword(s):  
Recombinant Proteins ◽  
Disulfide Bonds ◽  
Inclusion Bodies ◽  
Interleukin 2 ◽  
Oxidative Refolding

Cryo-electron microscopy of two-dimensional crystals of reduced type B botulinum neurotoxin

1992 ◽  
Vol 50 (1) ◽  
pp. 530-531
Author(s):  
P. F. Flicker ◽  
V.S. Kulkarni ◽  
J. P. Robinson ◽  
G. Stubbs ◽  
B. R. DasGupta
Keyword(s):  
Disulfide Bonds ◽  
Clostridium Botulinum ◽  
Structural Changes ◽  
Two Dimensional ◽  
Type B ◽  
Single Chain ◽  
Muscle Paralysis ◽  
Cryo Electron Microscopy ◽  
Disulfide Linkages ◽  
Intracellular Action

Botulinum toxin is a potent neurotoxin produced by Clostridium botulinum. The toxin inhibits release of neurotransmitter, causing muscle paralysis. There are several serotypes, A to G, all of molecular weight about 150,000. The protein exists as a single chain or or as two chains, with two disulfide linkages. In a recent investigation on intracellular action of neurotoxins it was reported that type B neurotoxin can inhibit the release of Ca++-activated [3H] norepinephrine only if the disulfide bonds are reduced. In order to investigate possible structural changes in the toxin upon reduction of the disulfide bonds, we have prepared two-dimensional crystals of reduced type B neurotoxin. These two-dimensional crystals will be compared with those of the native (unreduced) type B toxin.

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