• Mass Spectrometry Letters
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• v.1 no.1
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• pp.5-8
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• 2010

In the present study, collisionally-activated dissociation (CAD) experiments were performed under low energy collision conditions in six peptides containing a disulfide bond. Fragments produced as a result of the cleavage of a disulfide bond were obtained after CAD in four peptides (bactenecin, TGF-$\alpha$, cortistantin, and linearly linked peptide, Scheme 1) with basic amino acid residues. In contrast, the CAD analysis of two peptides with no basic residue (oxytocin and tocinoic acid) rarely produced fragments indicative of cleavage of a disulfide bond. These results are consistent with the mobile proton model suggested by the McLuckey and O'air groups (ref. 22 and 23); nonmobile protons sequestered at basic amino acid residues appear to promote the cleavage of disulfide bonds.

• BMB Reports
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• v.32 no.1
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• pp.76-81
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• 1999

Dihydrolipoamide dehydrogenase (E3) belongs to the protein family of pyridine nucleotide-disulfide oxidoreductases, including glutathione reductase (GR). The two subunits of human GR are covalently linked by an intersubunit disulfide bond between the pair of the Cys-90 residues. The corresponding residue (Ser-79) in human E3 was substituted to Cys using site-directed mutagenesis. The mutant was expressed in Escherichia coli and highly purified using an affinity column. About 40% of the mutants formed a spontaneous intersubunit disulfide bond linkage. This result implies that Ser-79 and possibly surrounding residues constitute one of the several intersubunit contact regions in human E3. It provides another good piece of evidence for the predicted high degree of the structural homology between human E3 and GR. Spectroscopic studies indicate conformational changes in the mutant.

• Bulletin of the Korean Chemical Society
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• v.26 no.4
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• pp.553-557
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• 2005

The formation of striped phases of unsymmetric hexyl octadecyl disulfide ($CH_3(CH_2)_5SS(CH_2)_{17}CH_3$, HOD) and 1-hydroxyundecyl octadecyl disulfide ($CH_3(CH_2)_{17}SS(CH_2)_{11}$OH, HUOD) on Au(111) and graphite has been investigated by scanning tunneling microscopy (STM) to understand the self-assembly processes of dialkyl disulfides. STM imaging clearly shows the formation of striped phases having corrugation periodicities that are nearly consistent with the molecular length of alkanethiolate moieties formed after the S-S bond cleavage of dialkyl disulfide on a gold surface. On the other hand, self-assembled monolayers (SAMs) of dialkyl disulfides on a graphite surface displayed long-range, well-ordered monolayers with one striped pattern that shows periodicity as a function of molecular length via nondissociative adsorption. From a nonoscopic viewpoint, we have clearly demonstrated that dialkyl disulfide SAMs on gold form via S-S bond cleavage of disulfide.

• Journal of Life Science
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• v.18 no.4
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• pp.585-589
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• 2008

Chronophin is a phosphatase responsible for the dephosphorylation of cofilin, which regulates the rearrangement of actin cytoskeleton. It is also known as a phosphatase for pyrodoxal 5'-phosphate (PLP), an active form of vitamin $B_6$, and maintains the level of PLP in the cytoplasm. Since this phosphatase belongs to a HAD subfamily containing a cap domain, it is expected to undergo a conformational change for the binding of a substrate. However, the crystal structure of chronophin has a disulfide bridge between the cap and core domains preventing a movement of the cap domain against the core domain. It is possible that the disulfide bond between C91 and C221 was formed by an oxidation during the crystallization. Here, we obtained chronophin crystals under a reduced condition and determined the crystal structure. This reduced chronophin does not contain a disulfide bridge and shows a closed conformation like the oxidized form. It implies that an active chronophin binds its substrate under the closed conformation without the disulfide bond and shows a high substrate specificity in the cell.

• Archives of Pharmacal Research
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• v.28 no.5
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• pp.561-565
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• 2005

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of sulfate group from a phenyl sulfate ester to a phenolic acceptor. The promoter region and the transcripti on start sites of Enterobacter amnigenus astA have been determined by primer extension analysis. Northern blot analysis resolved two mRNA species with lengths of 3.3 and 2.0 kb, which correspond to the distances between the transcriptional initiation sites and the two inverted repeat sequences (IRSs). By length, the 3.3 kb RNA could comprise the three-gene (astA with dsbA and dsbB) operon. ASST has three highly conserved cysteine residues. Reducing and non-reducing SDS-PAGE and activity staining showed that disulfide bond is needed for the activity of the enzyme. To identify the cysteine residues responsible for the disulfide bond formation, a series of Cys to Ser mutants has been constructed and the enzymatic activity was measured. Based on the results, we assumed that the first cysteine (Cys349) might be involved in disulfide bond mainly with the second cysteine (Cys445) and result in active conformation.

• Korean Chemical Engineering Research
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• v.55 no.1
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• pp.27-33
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• 2017

The determination of disulfide bonds is important for comprehensive understanding of the chemical structure of protein. So far, many strategies for the disulfide bond analysis have been suggested in terms of speed and sensitivity. However, most of these strategies have not considered free thiol residues in the target protein in the process of determining the disulfide bond. We suggested the strategy which was composed of four steps for the identification of disulfide bonds; the first step was the prediction of possible disulfide bonds, the second step was the determination of free cysteine residues, the third step was the analysis of disulfide bond using a high-resolution mass spectrometry, and the final step was the determination of disulfide bonds based on the comprehensive verification. In this study, we performed the characterization of disulfide bonds for the recombinant protein (HRPE1), where 1 and 5 inter- and intra-chain disulfide bonds were identified, respectively.

• Bulletin of the Korean Chemical Society
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• v.34 no.6
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• pp.1903-1905
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• 2013

• Biotechnology and Bioprocess Engineering:BBE
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• v.1 no.1
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• pp.13-17
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• 1996

The protein disulfide isomerase(PDI) reaction kinetics has been studied to evaluate its effect on the monoclonal antibody(MAb) refolding and assembly which accompanies disulfide bond formation The MAb in vitro assembly experiments showed that the assembly rate of heavy and light chains can be greatly enhanced in the presence of PDI as compared to the rate of assembly obtained by the air-oxidation. The reassembly patterns of MAb intermediates were identical for both with and without PDI, suggesting that the PDI does not determine the MAb assembly pathway, but rather facilitates the rate of MAb assembly by promoting PDI catalyzed disulfide bond formation. The effect of growth rate on PDI activities for MAb production has also been examined by using continuous culture system. The specific MAb productivity of hybridoma cells decreased as the growth rate increased. However, PDI activities were nearly constant for a wide range of growth rates except very high growth rate, indicating that no direct correlation between PDI activity and specific MAb productivity exists.

• Molecules and Cells
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• v.27 no.6
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• pp.673-680
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• 2009

Conversion of the normal soluble form of prion protein, PrP ($PrP^C$), to proteinase K-resistant form ($PrP^{Sc}$) is a common molecular etiology of prion diseases. Proteinase K-resistance is attributed to a drastic conformational change from ${\alpha}$-helix to ${\beta}$-sheet and subsequent fibril formation. Compelling evidence suggests that membranes play a role in the conformational conversion of PrP. However, biophysical mechanisms underlying the conformational changes of PrP and membrane binding are still elusive. Recently, we demonstrated that the putative transmembrane domain (TMD; residues 111-135) of Syrian hamster PrP penetrates into the membrane upon the reduction of the conserved disulfide bond of PrP. To understand the mechanism underlying the membrane insertion of the TMD, here we explored changes in conformation and membrane binding abilities of PrP using wild type and cysteine-free mutant. We show that the reduction of the disulfide bond of PrP removes motional restriction of the TMD, which might, in turn, expose the TMD into solvent. The released TMD then penetrates into the membrane. We suggest that the disulfide bond regulates the membrane binding mode of PrP by controlling the motional freedom of the TMD.

• Journal of Microbiology and Biotechnology
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• v.34 no.5
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• pp.1126-1134
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• 2024

The production of disulfide bond-containing recombinant proteins in Escherichia coli has traditionally been done by either refolding from inclusion bodies or by targeting the protein to the periplasm. However, both approaches have limitations. Two broad strategies were developed to allow the production of proteins with disulfide bonds in the cytoplasm of E. coli: i) engineered strains with deletions in the disulfide reduction pathways, e.g. SHuffle, and ii) the co-expression of oxidative folding catalysts, e.g. CyDisCo. However, to our knowledge, the relative effectiveness of these strategies has not been properly evaluated. Here, we systematically compare the purified yields of 14 different proteins of interest (POI) that contain disulfide bonds in their native state when expressed in both systems. We also compared the effects of different background strains, commonly used promoters, and two media types: defined and rich autoinduction. In rich autoinduction media, POI which can be produced in a soluble (non-native) state without a system for disulfide bond formation were produced in higher purified yields from SHuffle, whereas all other proteins were produced in higher purified yields using CyDisCo. In chemically defined media, purified yields were at least 10x higher in all cases using CyDisCo. In addition, the quality of the three POI tested was superior when produced using CyDisCo.