SEARCH · Search PubMed

Results for “Disulfides”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,387 records · Page 77Linked to original sources

PubMed2005

Structural determinants of substrate access to the disulfide oxidase Erv2p.

Erv2p is a small, dimeric FAD-dependent sulfhydryl oxidase that generates disulfide bonds in the lumen of the endoplasmic reticulum. Mutagenic and structural studies suggest that Erv2p uses an internal thiol-transfer relay between the FAD-proximal active site cysteine pair (Cys121-Cys124) and a second cysteine pair (Cys176-Cys178) located in a flexible, substrate-accessible C-terminal tail of the adjacent dimer subunit. Here, we demonstrate that Cys176 and Cys178 are the only amino acids in the tail region required for disulfide transfer and that their relative positioning within the tail peptide is important for activity. However, intragenic suppressor mutations could be isolated that bypass the requirement for Cys176 and Cys178. These mutants were found to disrupt Erv2p dimerization and to increase the activity of Erv2p for thiol substrates such as glutathione. We propose that the two Erv2p subunits act together to direct the disulfide transfer to specific substrates. One subunit provides the catalytic domain composed of the active site cysteine residues and the FAD cofactor, while the second subunit appears to have two functions: it facilitates disulfide transfer to substrates via the tail cysteine residues, while simultaneously shielding the active site cysteine residues from non-specific reactions.

Binding Sites↗

PubMed2006

Influence of the internal disulfide bridge on the folding pathway of the CL antibody domain.

Disulfide bridges are one of the most important factors stabilizing the native structure of a protein. Whereas the basis for their stabilizing effect is well understood, their role in a protein folding reaction still seems to require further attention. We used the constant domain of the antibody light chain (C(L)), a representative of the ubiquitous immunoglobulin (Ig)-superfamily, to delineate the kinetic role of its single buried disulfide bridge. Independent of its redox state, the monomeric C(L) domain adopts a typical Ig-fold under native conditions and does not retain significant structural elements when unfolded. Interestingly, its folding pathway is strongly influenced by the disulfide bridge. The more stable oxidized protein folds via a highly structured on-pathway intermediate, whereas the destabilized reduced protein populates a misfolded off-pathway species on its way to the native state. In both cases, the formation of the intermediate species is shown to be independent of the isomerization state of the Tyr(141)-Pro(142) bond. Our results demonstrate that the internal disulfide bridge in an antibody domain restricts the folding pathway by bringing residues of the folding nucleus into proximity thus facilitating the way to the native state.

Animals↗

PubMed2006

Nitro-thiocyanobenzoic acid (NTCB) reactivity of cysteines beta100 and beta110 in porcine luteinizing hormone: metastability and hypothetical isomerization of the two disulfide bridges of its beta-subunit seatbelt.

Luteinizing hormone (LH) like all other glycoprotein hormones is composed of two dissimilar subunits, alpha and beta, that are non-covalently associated. The heterodimer is stabilized by a region of the beta-subunit called the "seatbelt" because it wraps around the alpha-subunit and it is fastened by a disulfide bridge between cysteines beta26 and beta110. Although all 22 cysteines of porcine LH (pLH) are engaged in disulfide bridges, we previously showed that the free cysteine-specific reagent NTCB could react with pLH: it slowly cyanylated two cysteines in pLH and there was a close relationship between NTCB reaction with pLH and association/dissociation kinetics of its subunits. Therefore, cysteines beta26 and beta110 were considered as the best candidates for NTCB reaction. In order to identify the NTCB-reactive cysteines in pLH we have performed a mass spectroscopic analysis of the peptides released after mild basic hydrolysis of S-cyanylated pLH and its subunits. Only cysteines beta100 and beta110 were found to react with NTCB. Since these residues are not linked by a disulfide bridge in the crystallographic 3D structure of gonadotropins, it is proposed that their respective counterparts (Cysbeta93 and beta26) do not react with NTCB either because they are shielded from solvent or because they form a transient bridge. In the first hypothesis, both seatbelt bridges would be independently metastable; in the second one, a fast reversible isomerization between bridges beta26-beta110 and beta93-beta100 would occur. Such a reaction could be catalyzed by the previously recognized intrinsic protein disulfide isomerase (PDI) activity of gonadotropins.

Amino Acid Sequence↗

PubMed2005

Disruption of disulfide bond formation alters the trafficking of prothyrotropin releasing hormone (proTRH)-derived peptides.

Rat prothyrotropin releasing hormone (proTRH) is processed in the regulated secretory pathway (RSP) of neuroendocrine cells yielding five TRH peptides and several non-TRH peptides. It is not understood how these peptides are targeted to the RSP. We show here that a disulfide bond in the carboxy-terminus of proTRH plays an important role in the trafficking of this prohormone. Recombinant proTRH was observed to migrate faster on a native gel when treated with dithiothreitol (DTT) suggesting the presence of a disulfide bond. In vitro disulfide bond formation was prevented either by DTT treatment or by mutating cysteines 213 and 219 to glycines. In both cases the peptides derived from these mutants exhibited increased constitutive release and processing defects when expressed in AtT20 cells, a neuroendocrine cell line used in our prior studies on proTRH processing. Immunocytochemistry revealed that wild-type proTRH and mutant proTRH localized in a punctate pattern typical of proteins sorted to the regulated secretory pathway. These data suggest that the proposed disulfide bond of proTRH is involved in sorting of proTRH-derived peptides and in their retention within maturing secretory granules. This is the first evidence of structural motifs being important for the sorting of proTRH.

Amino Acid Motifs↗

PubMed2006

Role of the Cys18-Cys274 disulfide bond and of the third extracellular loop in the constitutive activation and internalization of angiotensin II type 1 receptor.

An insertion of residues in the third extracellular loop and a disulfide bond linking this loop to the N-terminal domain were identified in a structural model of a G-protein coupled receptor specific to angiotensin II (AT1 receptor), built in homology to the seven-transmembrane-helix bundle of rhodopsin. Both the insertion and the disulfide bond were located close to an extracellular locus, flanked by the second extracellular loop (EC-2), the third extracellular loop (EC-3) and the N-terminal domain of the receptor; they contained residues identified by mutagenesis studies to bind the angiotensin II N-terminal segment (residues D1 and R2). It was postulated that the insertion and the disulfide bond, also found in other receptors such as those for bradykinin, endothelin, purine and other ligands, might play a role in regulating the function of the AT1 receptor. This possibility was investigated by assaying AT1 forms devoid of the insertion and with mutations to Ser on both positions of Cys residues forming the disulfide bond. Binding and activation experiments showed that abolition of this bond led to constitutive activation, decay of agonist binding and receptor activation levels. Furthermore, the receptors thus mutated were translocated to cytosolic environments including those in the nucleus. The receptor form with full deletion of the EC-3 loop residue insertion, displayed a wild type receptor behavior.

Amino Acid Sequence↗

PubMed2004

Evidence for disulfide involvement in the regulation of intramolecular autolytic processing by human adamalysin19/ADAM19.

Human adamalysin 19 (a disintegrin and metalloproteinase 19, hADAM19) is activated by furin-mediated cleavage of the prodomain followed by an autolytic processing within the cysteine-rich domain at Glu586-Ser587, which occurs intramolecularly, producing an NH2 terminal fragment (N-fragment) associated with its COOH-terminal fragment (C-fragment), most likely through disulfide bonds. When stable Madin-Darby canine kidney (MDCK) transfectants overexpressing soluble hADAM19 were treated with dithiothreitol (DTT) or with media at pH 6.5, 7.5, or 8.5, the secretion and folding of the enzyme were not affected. Autolytic processing was blocked by DTT and pH 6.5 media, which favor disulfide reduction, but was increased by pH 8.5 media, which promotes disulfide formation. Cys605, Cys633, Cys639, and Cys643 of the C-fragment appear to be partially responsible for the covalent association between the C-fragment and the N-fragment. A new autolytic processing site at Lys543-Val544 was identified in soluble mutants when these cysteine residues were individually mutated to serine residues. Shed fragments were also detectable in the media from MDCK cells stably expressing the full-length Cys633Ser mutant. Ilomastat/GM6001 inhibited hADAM19 with an IC50 of 447 nM, but scarcely affected the shedding process. The cysteine-rich domain likely forms disulfide bonds to regulate the autolytic processing and shedding of hADAM19.

ADAM Proteins↗

PubMed2002

Sulfhydryl oxidases: emerging catalysts of protein disulfide bond formation in eukaryotes.

Members of the Quiescin-sulfhydryl oxidase (QSOX) family utilize a thioredoxin domain and a small FAD-binding domain homologous to the yeast ERV1p protein to oxidize sulfhydryl groups to disulfides with the reduction of oxygen to hydrogen peroxide. QSOX enzymes are found in all multicellular organisms for which complete genomes exist and in Trypanosoma brucei, but are not found in yeast. The avian QSOX is the best understood enzymatically: its preferred substrates are peptides and proteins, not monothiols such as glutathione. Mixtures of avian QSOX and protein disulfide isomerase catalyze the rapid insertion of the correct disulfide pairings in reduced RNase. Immunohistochemical studies of human tissues show a marked and highly localized concentration of QSOX in cell types associated with heavy secretory loads. Consistent with this role in the formation of disulfide bonds, QSOX is typically found in the cell in the endoplasmic reticulum and Golgi and outside the cell. In sum, this review suggests that QSOX enzymes play a significant role in oxidative folding of a large variety of proteins in a wide range of multicellular organisms.

Amino Acid Sequence↗

PubMed2003

Disulfide bonds convert small heat shock protein Hsp16.3 from a chaperone to a non-chaperone: implications for the evolution of cysteine in molecular chaperones.

Molecular chaperones mainly function in assisting newly synthesized polypeptide folding and protect non-native proteins from aggregation, with known structural features such as the ability of spontaneous folding/refolding and high conformational flexibility. In this report, we verified the assumption that the lack of disulfide bonds in molecular chaperones is a prerequisite for such unique structural features. Using small heat shock protein (one sub-class of chaperones) Hsp16.3 as a model system, our results show the following: (1) Cysteine-free Hsp16.3 wild type protein can efficiently exhibit chaperone activity and spontaneously refold/reassemble with high conformational flexibility. (2) Whereas Hsp16.3 G89C mutant with inter-subunit disulfide bonds formed seems to lose the nature of chaperone proteins, i.e., under stress conditions, it neither acts as molecular chaperone nor spontaneously refolds/reassembles. Structural analysis indicated that the mutant exists as an unstable molten globule-like state, which incorrectly exposes hydrophobic surfaces and irreversibly tends to form aggregates that can be suppressed by the other molecular chaperone (alpha-crystallin). By contrast, reduction of disulfide bond in the Hsp16.3 G89C mutant can significantly recover its character as a molecular chaperone. In light of these results, we propose that disulfide bonds could severely disturb the structure/function of molecular chaperones like Hsp16.3. Our results might not only provide insights into understanding the structural basis of chaperone upon binding substrates, but also explain the observation that the occurrence of cysteine in molecular chaperones is much lower than that in other protein families, subsequently being helpful to understand the evolution of protein family.

Amino Acid Sequence↗

PubMed1990

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis.

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.

Amino Acid Sequence↗

PubMed1988

Demonstration of a 1-3 disulfide bond in a synthetic nonapeptide derived from the signal sequence and N-terminus of human gamma-interferon.

The nonapeptide Cys-Tyr-Cys-Gln-Asp-Pro-Tyr-Val-Lys was prepared by solid-phase peptide synthesis under oxidizing conditions. Fast atom bombardment mass spectrometric analysis of the untreated molecule produced an ion consistent with a structure involving an intramolecular disulfide bond between Cys(1) and Cys(3). Mass spectra of the peptide after treatment with 2-mercaptoethanol gave signals corresponding to the reduced disulfide form of the peptide and to a mixed disulfide of the peptide with 2-mercaptoethanol. Molecular mechanics calculations of the conformation of the 11-membered ring formed by disulfide bond closure predicted a discrete, low-energy structure resembling the locus of a gamma turn. We hypothesize that this structure may be important in the recognition and cleavage of the signal sequence of the parent molecule.

Chromatography, High Pressure Liquid↗

PubMed2000

Effects of disulfide bonds formed during isolation process on the structure of the postsynaptic density.

The biochemical, morphological and structural properties of rat postsynaptic densities (PSDs) isolated under conditions where disulfide bond formation was allowed or curtailed were studied here. Biochemical analyses revealed that the isolated PSDs were composed by a similar set of proteins regardless of the differences in their isolation processes. The PSDs isolated under the conditions where disulfide bond formation was curtailed were more easily dissociated by treatments with urea, guanidine hydrochloride and deoxycholate than the PSDs isolated under conditions where disulfide bond formation was allowed. Consistently, the structure of the PSDs isolated under the former condition appeared to be more fragmented than those isolated under the latter condition, as revealed by electron microscopy. The results indicate that the disulfide bonds formed during the isolation process significantly tighten the PSD structure and further suggest that the PSD in vivo is a protein aggregate whose constituent proteins be held together primarily by non-covalent interactions.

Animals↗

PubMed1995

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen.

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Raman spectroscopy of calf lens gamma-II crystallin: direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione.

This study presents Raman spectra of calf lens gamma-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in gamma-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S--S bond stretching vibration from the mixed disulfides at 510 cm-1, which is very similar to the 508 cm-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J.F.R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A., 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm-1, which is tentatively attributed to the Hg--S stretching vibration of the mercaptide complex. The S--H vibration region (2500-2700 cm-1) exhibits two resolved peaks at 2562 and 2580 cm-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S--H vibration at 2580 cm-1.

Animals↗

PubMed2000

Formation of native structure by intermolecular thiol-disulfide exchange reactions without oxidants in the folding of bovine pancreatic ribonuclease A.

It has been shown previously that the oxidative folding of bovine pancreatic ribonuclease A proceeds through parallel pathways with two major native-like three-disulfide (3S) intermediates. We show here that, under some conditions, the native disulfide bonds can also be regenerated through disproportionation reactions; in other words, the protein can serve as its own redox reagent. The results also show that disulfide species of the unstructured 3S ensemble have a strong propensity to participate in intermolecular interactions. These interactions are favored at high protein concentration, temperature and pH, and lead to formation of the native structure during disulfide reshuffling in the rate-determining step.

Animals↗

PubMed2002

The unfolding mechanism and the disulfide structures of denatured lysozyme.

The mechanism of denaturation and unfolding of lysozyme has been characterized here using the method of disulfide scrambling. Under denaturing conditions (urea, guanidinium hydrochloride (GdmCl), guanidinium thiocyanate (GdmSCN), or elevated temperature) and in the presence of thiol initiator, lysozyme denatures by shuffling its four native disulfide bonds and converts to a mixture of fully oxidized scrambled isomers. To denature 50% of the native lysozyme requires 1.1 M of GdmSCN, 2.8 M of GdmCl and 7.4 M of urea, respectively. High temperature (75 degrees C) denatures the native lysozyme quantitatively within 20 min. Analysis by reversed-phase high-performance liquid chromatography reveals that urea and GdmCl denatured lysozyme comprise a single predominant disulfide isomer, designated as X-lysozyme-a, regardless of the concentration of the denaturant. X-Lysozyme-a was shown to adopt the beads-form structure with its four disulfide bonds formed by four consecutive pairs of cysteines (Cys6-Cys30, Cys64-Cys76, Cys80-Cys94, Cys115-Cys127). The conspicuous absence of partially structured unfolding intermediates of lysozyme contrasts to that found in the case of alpha-lactalbumin and accounts for the widely observed two-stage mechanism of lysozyme unfolding.

Animals↗

PubMed1999

Disulfide structure of the pheromone binding protein from the silkworm moth, Bombyx mori.

Disulfide bond formation is the only known posttranslational modification of insect pheromone binding proteins (PBPs). In the PBPs from moths (Lepidoptera), six cysteine residues are highly conserved at positions 19, 50, 54, 97, 108 and 117, but to date nothing is known about their respective linkage or redox status. We used a multiple approach of enzymatic digestion, chemical cleavage, partial reduction with Tris-(2-carboxyethyl)phosphine, followed by digestion with endoproteinase Lys-C to determine the disulfide connectivity in the PBP from Bombyx mori (BmPBP). Identification of the reaction products by on-line liquid chromatography-electrospray ionization mass spectrometry (LC/ESI-MS) and protein sequencing supported the assignment of disulfide bridges at Cys-19-Cys-54, Cys-50-Cys-108 and Cys-97-Cys-117. The disulfide linkages were identical in the protein obtained by periplasmic expression in Escherichia coli and in the native BmPBP.

Amino Acid Sequence↗

PubMed1998

Rapid verification of disulfide linkages in recombinant human growth hormone by tandem column tryptic mapping.

An automated tryptic mapping method was developed for characterization of disulfide linkages in recombinant human growth hormone (rhGH). The hormone was trypsin digested and the peptide fragments concentrated by eluting rhGH through an immobilized trypsin column and transferring the peptides directly to a reversed-phase liquid chromatography (RP-LC) column where they were collected. Reaction time was controlled by the flow-rate. Following tryptic digestion of a sample, the immobilized enzyme column was uncoupled from the flow train by a switching valve and the RP-LC column eluted with a solvent gradient ranging from 0.1% trifluoroacetic acid (TFA) with 1% acetonitrile (ACN) to ACN with 0.1% TFA and 5% water. This two-step mapping process was achieved within 2 h on both native and reduced rhGH samples. The chromatographic elution position and mass spectra matrix-assisted laser desorption ionization time-of-flight mass spectrometry of native rhGH and sulfur-containing peptides were determined with standards. Standards of the individual sulfhydryl (-SH) containing peptides and all possible disulfide linked peptides that could result from coupling the -SH peptides in disulfide linkages were obtained by synthesis and chromatographic purification. This approach allowed the chromatographic elution position of all possible mismatched disulfide containing peptides to be established and samples of rhGH to be examined for improper folding.

Amino Acid Sequence↗

PubMed1999

Eliminating disulfide exchange during glutamyl endopeptidase digestion of native protein.

Numerous advantages of using immobilized enzymes over free-solution protein digests have been cited in the literature. This investigation examines both the rate of hydrolysis and the extent of disulfide bond exchange in disulfide bridged dipeptide fragments formed during proteolysis of native protein. Glutamyl endopeptidase as both an immobilized enzyme and in free solution was used in these studies. It was found that extensive hydrolysis of insulin was achieved in 2 min with immobilized enzyme cartridges operated in the stopped-flow mode orders. This is orders of magnitude faster than was seen in free solution. Other advantages ranging from ease of use and reduction in sample size to the potential for automation were also noted with the immobilized enzyme cartridge. Normal free-solution proteolysis generally requires 12-24 h, based on the lower enzyme-to-substrate ratio in solution. A disturbing feature noted in these lengthy free-solution reactions was the tendency to form disulfide bridged peptide artifacts. This could lead to the erroneous conclusion that disulfide bonding in a sample was not that of the native protein. It is concluded that the advantage of immobilized enzymes over free-solution reactions will be most important in the pharmaceutical industry where proteolytic fragment "fingerprinting" of recombinant proteins is being used to confirm structure.

Amino Acid Sequence↗