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PubMed1994

Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3.

The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S. DHHS, NIH publication No. 91-3242, 1991). This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.

ADP Ribose Transferases↗
PubMed1994

Thiamine disulfide as a potent inhibitor of human immunodeficiency virus (type-1) production.

Thiol and disulfide compounds were tested as an anti-HIV drug against transactivator (Tat)-mediated transactivation of HIV-1. Of all the compounds tested, thiamine disulfide, alpha-lipoic acid, and N-acetycysteine significantly depressed HIV-1 Tat activity. Thiamine disulfide alone in these compounds possessing anti-HIV-Tat activity markedly inhibited production of progeny HIV-1 in acute and chronic HIV-1-infected CEM at nontoxic concentrations of 500-1000 microM. Thiamine disulfide (500 microM) blocked 99.7% of HIV-1 production after 96 hr culture in acute HIV-1 (LAV-1) infection (m.o.i. = 0.002), whereas it inhibited 90-98% of HIV-1 production in chronic-infected cells (CEM/LAV-1, H9/MN, and Molt-4/IIIB). The results suggest that thiamine disulfide may be important for AIDS chemotherapy.

Acetylcysteine↗
PubMed1999

NMR structure of Escherichia coli glutaredoxin 3-glutathione mixed disulfide complex: implications for the enzymatic mechanism.

Glutaredoxins (Grxs) catalyze reversible oxidation/reduction of protein disulfide groups and glutathione-containing mixed disulfide groups via an active site Grx-glutathione mixed disulfide (Grx-SG) intermediate. The NMR solution structure of the Escherichia coli Grx3 mixed disulfide with glutathione (Grx3-SG) was determined using a C14S mutant which traps this intermediate in the redox reaction. The structure contains a thioredoxin fold, with a well-defined binding site for glutathione which involves two intermolecular backbone-backbone hydrogen bonds forming an antiparallel intermolecular beta-bridge between the protein and glutathione. The solution structure of E. coli Grx3-SG also suggests a binding site for a second glutathione in the reduction of the Grx3-SG intermediate, which is consistent with the specificity of reduction observed in Grxs. Molecular details of the structure in relation to the stability of the intermediate and the activity of Grx3 as a reductant of glutathione mixed disulfide groups are discussed. A comparison of glutathione binding in Grx3-SG and ligand binding in other members of the thioredoxin superfamily is presented, which illustrates the highly conserved intermolecular interactions in this protein family.

Amino Acid Sequence↗
PubMed1990

Fluorescence lifetime and rotational correlation time of bovine serum albumin-sodium dodecyl sulfate complex labeled with 1-dimethylaminonaphthalene-5-sulfonyl chloride: effect of disulfide bridges in the protein on these fluorescence parameters.

A fluorescent dye, 1-dimethylaminonaphthalene-5-sulfonyl chloride, was used to label bovine serum albumin (BSA), intact and disulfide bridges-cleaved. The fluorescence lifetime and fluorescence anisotropy of the adducts in sodium dodecyl sulfate (SDS) solutions were studied by the nanosecond fluorescence depolarization method. The volume of equivalent sphere (Ve) was calculated to be 2.1 x 10(-19) cm3 for BSA with the intact disulfide bridges from the rotational correlation time. The value of Ve was 4.4 x 10(-19) cm3 for the disulfide bridges-cleaved BSA. With an increase in SDS concentration, the rotational correlation time of the intact BSA became longer, while that of the disulfide bridges-cleaved BSA became shorter. This suggests that upon the binding of SDS, the total volume of the intact BSA increases while the expanded state of the protein, caused by the cleavage of the disulfide bridges, becomes compact.

Circular Dichroism↗
PubMed1992

Protein sulfhydryls are protected from irreversible oxidation by conversion to mixed disulfides.

Protein mixed thioselenides formed by reaction of sarcoplasmic reticulum (SR) with diselenide biradical spin labels were quantified by ESR. Whereas the reaction of SR membranes with the diselenide spin label led to a large ESR signal of the unbound monoselenide at equilibrium, treatment of the reaction mixture with a few millimolar hydrogen peroxide converted all of the nitroxides to protein-bound thioselenides. This technique of spin-labeling protein thiols avoids the need to remove unreacted spin labels. The bound spin labels were removable by reduction with excess mercaptoethanol, indicating a specific and reversible labeling of protein thiols. SR that had been extensively labeled with the diselenide spin label was resistant to ATPase inactivation by potent oxidants that arise when myoglobin reacts with hydroperoxides. Unmodified SR lost all activity within 10 min of exposure to either 1 mM tert-butyl hydroperoxide in the presence of 200 microM equine myoglobin or to 100 mM hydrogen peroxide in the absence of myoglobin. In both cases the loss of activity could not be reversed by subsequent treatment with mercaptoethanol. On the other hand, membranes that had been extensively treated with the diselenide spin label and were then subjected to these peroxide treatments were fully active after mercaptoethanol-mediated cleavage of the thioselenides. ESR analysis of spin-labeled SR showed no detectable oxidative cleavage of the thioselenide bonds. Sodium dodecyl sulfate gel electrophoresis showed that peroxide-mediated crosslinking of ATPase observed in unmodified SR membranes did not occur in the diselenide-modified SR membranes. Only limited protection was observed when SR pretreated with glutathione disulfide was incubated with hydroperoxides. In this case, however, the degree of protection was greatly increased when the reaction with glutathione disulfide was carried out in the presence of the supernatant of centrifuged rat liver homogenate, consistent with an acceleration of mixed disulfide formation by a factor tentatively identified as thiol transferase. It is concluded that conversion of protein thiol residues to either thioselenides or mixed disulfides confers protection against irreversible peroxide-dependent oxidation. We suggest that mixed disulfide formation by thiol transferase activity may help protect protein thiols from irreversible oxidation by heme-activated hydroperoxides.

Adenosine Triphosphatases↗
PubMed1987

Mode of disulfide bond formation of a heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli.

To determine the modes of three disulfide linkages in the heat-stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli, we synthesized STh(6-18), which consists of 13 amino acid residues and has the same intramolecular disulfide linkages as native STh [(1985) FEBS Lett. 181, 138-142], by stepwise and selective formation of disulfide bonds using different types of removable protecting groups for the Cys residues. Synthesis of the peptide with different modes of disulfide bond formation provided three peptides consistent with standard STh(6-18) in their physicochemical and biological properties, thereby indicating that the disulfide bonds in STh(6-18) are Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Thr-Gly-Cys.

Animals↗
PubMed2006

The roles of surface loop insertions and disulfide bond in the stabilization of thermophilic WF146 protease.

Thermophilic WF146 protease possesses four surface loop insertions and a disulfide bond, resembling its psychrophilic (subtilisins S41 and S39) and mesophilic (subtilisins SSII and sphericase) homologs. Deletion of the insertion 3 (positions 193-197) or insertion 4 (positions 210-221) of WF146 protease resulted in a significant decrease of the enzyme stability. In addition, substitution of the residues Pro211 and Ala212 or residue Glu221 which localized in the vicinity of a Ca(2+) binding site of the enzyme by the corresponding residues in subtilisin S41 remarkably reduced the half-life of the enzyme at 70 degrees C, suggesting that the three residues contributed to the thermostability of the enzyme, probably by enhancing the affinity of enzyme to Ca(2+). In the presence of dithiothreitol, the WF146 protease suffered excessive autolysis, indicating that the Cys52-Cys65 disulfide bond played a critical role in stabilizing the WF146 protease against autolysis. The autolytic cleavage sites of the WF146 protease were identified to locate between residues Asn63-Gly64 and Cys65-Ala66 by N-terminal amino acid analysis of the autolytic product. It was noticed that the effect of the autolytic cleavage at Asn63-Gly64 could be compensated by the disulfide bond Cys52-Cys65 under non-reducing condition, and the disulfide bond cross-linked autolytic product remained active. The apparent stabilization effect of the disulfide bond Cys52-Cys65 in the WF146 protease might provide a rational basis for improving the stability of subtilase against autolysis by protein engineering.

Amino Acid Sequence↗
PubMed2004

Structure of Ero1p, source of disulfide bonds for oxidative protein folding in the cell.

The flavoenzyme Ero1p produces disulfide bonds for oxidative protein folding in the endoplasmic reticulum. Disulfides generated de novo within Ero1p are transferred to protein disulfide isomerase and then to substrate proteins by dithiol-disulfide exchange reactions. Despite this key role of Ero1p, little is known about the mechanism by which this enzyme catalyzes thiol oxidation. Here, we present the X-ray crystallographic structure of Ero1p, which reveals the molecular details of the catalytic center, the role of a CXXCXXC motif, and the spatial relationship between functionally significant cysteines and the bound cofactor. Remarkably, the Ero1p active site closely resembles that of the versatile thiol oxidase module of Erv2p, a protein with no sequence homology to Ero1p. Furthermore, both Ero1p and Erv2p display essential dicysteine motifs on mobile polypeptide segments, suggesting that shuttling electrons to a rigid active site using a flexible strand is a fundamental feature of disulfide-generating flavoenzymes.

Amino Acid Sequence↗
PubMed1996

Mixed disulfides from disulfiram inhibit the benzo[a]pyrene induced mutagenesis.

Some mixed disulfides derived from disulfiram and endogenous thiol compounds have been synthesized, biochemically characterized and their potential antigenotoxic effects have been proposed. The present study evaluated the mutagenic and antimutagenic specificities of two mixed disulfides using S. typhimurium reversion assay, namely S-(N,N-diethyldithiocarbamoyl)-N-acetylcysteine (AC-DDTC) and-L-glutathione (GS-DDTC). The two mixed disulfides were not mutagenic to Salmonella strains TA98 and TA100 in the presence or absence of S9 mixture. The increased number of revertants by benzo[a]pyrene (B(a)P) has been reduced to the control level by the preincubation with AC-DDTC or GS-DDTC. It was not due to the killing effect of B(a)P, mixed disulfides or B(a)P-disulfide mixture. The antimutagenic effect of AC-DDTC was more potent than that of GS-DDTC. These results indicate that AC-DDTC and GS-DDTC may have a role to play in reducing the risk of mutagenic effects of B(a)P.

Animals↗
PubMed2006

Structure of coenzyme A-disulfide reductase from Staphylococcus aureus at 1.54 A resolution.

Coenzyme A (CoASH) replaces glutathione as the major low molecular weight thiol in Staphylococcus aureus; it is maintained in the reduced state by coenzyme A-disulfide reductase (CoADR), a homodimeric enzyme similar to NADH peroxidase but containing a novel Cys43-SSCoA redox center. The crystal structure of S. aureus CoADR has been solved using multiwavelength anomalous dispersion data and refined at a resolution of 1.54 A. The resulting electron density maps define the Cys43-SSCoA disulfide conformation, with Cys43-S(gamma) located at the flavin si face, 3.2 A from FAD-C4aF, and the CoAS- moiety lying in an extended conformation within a cleft at the dimer interface. A well-ordered chloride ion is positioned adjacent to the Cys43-SSCoA disulfide and receives a hydrogen bond from Tyr361'-OH of the complementary subunit, suggesting a role for Tyr361' as an acid-base catalyst during the reduction of CoAS-disulfide. Tyr419'-OH is located 3.2 A from Tyr361'-OH as well and, based on its conservation in known functional CoADRs, also appears to be important for activity. Identification of residues involved in recognition of the CoAS-disulfide substrate and in formation and stabilization of the Cys43-SSCoA redox center has allowed development of a CoAS-binding motif. Bioinformatics analyses indicate that CoADR enzymes are broadly distributed in both bacterial and archaeal kingdoms, suggesting an even broader significance for the CoASH/CoAS-disulfide redox system in prokaryotic thiol/disulfide homeostasis.

Amino Acid Sequence↗
PubMed1997

Peptidylglycine alpha-hydroxylating monooxygenase: active site residues, disulfide linkages, and a two-domain model of the catalytic core.

Peptidylglycine alpha-hydroxylating monooxygenase (PHM) is a copper, ascorbate, and molecular oxygen dependent enzyme that catalyzes the first step leading to the C-terminal amidation of glycine-extended peptides. The catalytic core of PHM (PHMcc), refined to residues 42-356 of the PHM protein, was expressed at high levels in CHO (DG44) (dhfr-) cells. PHMcc has 10 cysteine residues involved in 5 disulfide linkages. Endoprotease Lys-C digestion of purified PHMcc under nonreducing conditions cleaved the protein at Lys219, indicating that the protein consists of separable N- and C-terminal domains with internal disulfide linkages, that are connected by an exposed linker region. Disulfide-linked peptides generated by sequential CNBr and pepsin treatment of radiolabeled PHMcc were separated by reverse phase HPLC and identified by Edman degradation. Three disulfide linkages occur in the N-terminal domain (Cys47-Cys186, Cys81-Cys126, and Cys114-Cys131), along with three of the His residues critical to catalytic activity (His107, His108, and His172). Two disulfide linkages (Cys227-Cys334 and Cys293-Cys315) occur in the C-terminal domain, along with the remaining two essential His residues (His242, His244) and Met314, thought to be essential in binding one of the two nonequivalent copper atoms. Substitution of Tyr79 or Tyr318 with Phe increased the Km of PHM for its peptidylglycine substrate without affecting the Vmax. Replacement of Glu313 with Asp increased the Km 8-fold and decreased the kcat 7-fold, again identifying this region of the C-terminal domain as critical to catalytic activity. Taking into account information on the copper ligands in PHM, we propose a two-domain model with a copper site in each domain that allows spatial proximity between previously described copper ligands and residues identified as catalytically important.

Animals↗
PubMed2000

Synthesis, structure, and H2O2-dependent catalytic functions of disulfide-bridged dicopper(I) and related thioether-copper(I) and thioether-copper(II) complexes.

A disulfide-bridged dicopper(I) complex, [Cu2(Py2SSPy2)](ClO4)2 (1) (Py2SSPy2 = bis(2-[N,N-bis(2-pyridylethyl)-amino]-1,1- dimethylethyl)disulfide), a thioether-copper(I) complex, [Cu(iPrSPy2)](ClO4) (2) (iPrSPy2 = N-(2-isopropylthio-2-methyl)propyl-N,N-bis-2-(2-pyridyl)ethylamine, and a thioether-copper(II) complex, [Cu-(PheSPy2)(H2O)](ClO4)2 (3) (PheSPy2 = N-(2-methyl-2-phenethylthio)propyl-N,N-bis-2-(2- pyridyl)ethylamine), were newly synthesized by the reactions of Cu(ClO4)2.6H2O with a thiol ligand of Py2SH (N,N-bis[2-(2-pyridyl)-ethyl]-1,1-dimethyl-2- mercaptoethylamine) and thioether ligands of iPrSPy2 and PheSPy2, respectively. For complexes 1 and 2, X-ray analyses were performed. Complex 1 crystallizes in the triclinic space group P1, and complex 2 crystallizes in the orthorhombic space group Pbca with the following unit cell parameters: for 1, a = 15.165 (3) A, b = 22.185 (4) A, c = 14.989 (3) A, alpha = 105.76 (1) degrees, beta = 90.82 (2) degrees, gamma = 75.23 (1) degrees, and Z = 2; for 2, a = 17.78 (2) A, b = 17.70 (1) A, c = 15.75 (1) A, and Z = 8. Complex 1 is the first structurally characterized example obtained by the redox reaction Cu(II) + RSH-->Cu(I) + RSSR and has two independent structures (1a, 1b) which mainly differ in S-S bond distances, Cu(I)...Cu(I) separations, and C-S-S-C dihedral angles of the disulfide units. The S-S bond distances of 2.088(7) A in 1a and 2.070(7) A in 1b are indicative of significant activation of the S-S bonds by the dicopper centers. Fragment molecular orbital (FMO) analyses and molecular orbital overlap population (MOOP) analyses based on the extended Hückel method clarify the preferable formation of the disulfide S-S bond in 1 rather than the formation of a thiolate-copper(II) complex within the Py2S- ligand framework. Catalytic functions of complexes 1-3 were investigated with peroxides (H2O2 and tBuOOH) as oxidants. Complex 1 catalyzed the selective oxidation of cyclohexane to cyclohexanol and mediated the cyclohexene epoxidation in the presence of H2O2. A transient dark green intermediate observed in the reaction of 1 with H2O2 is characterized by UV-vis, EPR, and resonance Raman spectroscopies, identifying it as a Cu(II)-OOH species, 1(OOH). The resonance Raman features of the nu(O-O) bands at 822 and 836 cm-1, which are red-shifted to 781 and 791 cm-1, respectively, upon introduction of H2(18)O2, are indicative of formation of two kinds of Cu-OOH species rather than the Fermi doublet and the significant weakening of the O-O bonds. These mechanistic studies demonstrate that by virtue of the electron-donating ability of the disulfide unit the Cu-OOH species can be actually activated for one-electron oxidation, which has been reported so far unfavorable for other vibrationally characterized Cu-OOH species.

Journal Article↗
PubMed2003

Formation and cleavage of aromatic disulfide radical anions.

The electron transfer (ET) to a series of para-substituted diaryl disulfides, having the general formula (X-C(6)H(4)S-)(2), has been studied. The X groups were selected as to have a comprehensive variation of the substituent effect, being X = NH(2), MeO, H, F, Cl, CO(2)Et, CN, and NO(2). The reduction was carried out experimentally, using N,N-dimethylformamide as the solvent, and by molecular orbital (MO) ab initio calculations. The ET was studied heterogeneously, by voltammetric reduction and convolution analysis, and homogeneously, by using electrogenerated radical anions as the solution electron donors. The reduction is dissociative, leading to the cleavage of the S-S bond in a stepwise manner. Both experimental approaches led us to estimate the E degrees and the intrinsic barrier values for the formation of the radical anions. Comparison of the independently obtained results allowed obtaining, for the first time, a quantitative description of the correlation between heterogeneous and homogeneous rate constants of ETs associated with significant inner reorganization energy. The experimental outcome was fully supported by the theoretical calculations, which provided information about the disulfide lowest unoccupied MOs (LUMOs) and singly occupied MO (SOMO), the bond dissociation energies, and the most significant structural modifications associated with radical anion formation. With disulfides bearing electron-donating or mildly electron-withdrawing groups, the inner reorganization is particularly large, which reflects the significant stretching of the S-S bond experienced by the molecule upon ET. The process entails formation of loose radical anion species in which the SOMO is heavily localized, as the LUMO, onto the frangible bond. As a consequence of the formation of these sigma-radical anions, the S-S bond energy of the latter is rather small and the cleavage rate constant is very large. With electron-withdrawing groups, the extent of delocalization of the SOMO onto the aryl system increases, leading to a decrease of the reorganization energy for radical anion formation. Interestingly, while the LUMO now has pi character, the actual reduction intermediate (and thus the SOMO) is still a sigma-type radical anion. With the nitro-substituted disulfide, very limited inner reorganization is required and a pi-radical anion initially forms. A nondissociative type intramolecular ET then ensues, leading to the formation of a new radical anion whose antibonding orbital has similar features as those of the SOMO of the other diaryl disulfides. Therefore, independently of the substituent, the actual S-S bond cleavage occurs in a quite similar way along the series investigated. The S-S bond cleavage rate, however, tends to decrease as the Hammett sigma increases, which would be in keeping with an increase of both the electronic and solvent reorganization energies.

Journal Article↗
PubMed2002

Synthesis and stable isotope dilution assay of ethanethiol and diethyl disulfide in wine using solid phase microextraction. Effect of aging on their levels in wine.

Ethanethiol and diethyl disulfide (DEDS) most often occurred at levels above their olfactive threshold in wines with nauseous sulfur-linked smells. As ethanethiol is very oxidizable and chemically reactive, a stable isotopic dilution analysis of both ethanethiol and its disulfide in wines using solid phase microextraction and GC-MS was developed. The latter involved the determination of the proportion of DEDS formed by oxidation of the thiol during the analysis conditions, which was obtained by the use of two differently labeled disulfide standards. An original synthesis of labeled ethanethiol standards in conditions minimizing oxidation was developed, and the corresponding labeled diethyl disulfides were obtained from these thiols. This analytical method was used to follow the levels of these sulfur compounds during aging in a young red wine spiked with ethanethiol and added with enological tannins, with or without oxygen addition. The total levels of these two sulfur compounds were shown to decrease steadily after 60 days of aging, up to 83%. The effect of oxygen sped this decrease, but the effect of enological tannins was very slight. Residual ethanethiol was detected in its disulfide form from approximately 36% in the nonoxygenated wines to 69% in the oxygenated samples.

Deuterium↗
PubMed2005

Transferable potentials for phase equilibria. 8. United-atom description for thiols, sulfides, disulfides, and thiophene.

An extension of the transferable potentials for phase equilibria united-atom (TraPPE-UA) force field to thiol, sulfide, and disulfide functionalities and thiophene is presented. In the TraPPE-UA force field, nonbonded interactions are governed by a Lennard-Jones plus fixed point charge functional form. Partial charges are determined through a CHELPG analysis of electrostatic potential energy surfaces derived from ab initio calculations at the HF/6-31g+(d,p) level. The Lennard-Jones well depth and size parameters for four new interaction sites, S (thiols), S(sulfides), S(disulfides), and S(thiophene), were determined by fitting simulation data to pure-component vapor-equilibrium data for methanethiol, dimethyl sulfide, dimethyl disulfide, and thiophene, respectively. Configurational-bias Monte Carlo simulations in the grand canonical ensemble combined with histogram-reweighting methods were used to calculate the vapor-liquid coexistence curves for methanethiol, ethanethiol, 2-methyl-1-propanethiol, 2-methyl-2-propanethiol, 2-butanethiol, pentanethiol, octanethiol, dimethyl sulfide, diethyl sulfide, ethylmethyl sulfide, dimethyl disulfide, diethyl disulfide, and thiophene. Excellent agreement with experiment is achieved, with unsigned errors of less than 1% for saturated liquid densities and less than 3% for critical temperatures. The normal boiling points were predicted to within 1% of experiment in most cases, although for certain molecules (pentanethiol) deviations as large as 5% were found. Additional calculations were performed to determine the pressure-composition behavior of ethanethiol+n-butane at 373.15 K and the temperature-composition behavior of 1-propanethiol+n-hexane at 1.01 bar. In each case, a good reproduction of experimental vapor-liquid equilibrium separation factors is achieved; both of the coexistence curves are somewhat shifted because of overprediction of the pure-component vapor pressures.

Journal Article↗
PubMedDate not supplied

Dissociations of disulfide-linked gaseous polypeptide/protein anions: ion chemistry with implications for protein identification and characterization.

Ion trap collisional activation of whole protein anions that contain disulfide bonds results in the cleavage of one of the bonds that comprises the disulfide linkage. The disulfide linkage can break at any of three possible locations, giving rise to several products with different partitioning of sulfur atoms. A facile second-generation dissociation occurs at the polypeptide backbone from products formed from cleavage of the nearest C-S bond of a disulfide linkage. This cleavage occurs exclusively at the N-terminal side of the cysteine residue, from which the C-S bond was cleaved, thereby yielding c and z-S type product ions. This secondary reaction is apparently a relatively low-energy reaction with relatively high entropy requirements because it is not observed to be a major process under beam-type collisional activation conditions, but is a major process under ion trap collisional activation conditions. The specificity of this cleavage, as well as the ability to distinguish it from other cleavages by the sulfur atom distribution, make it useful for the identification of unknown proteins via database searching. Furthermore, the pattern of disulfide cleavages can be useful in providing information about the location of post-translational modifications. Examples using bovine pancreatic trypsin inhibitor and ribonuclease A and B are given to illustrate these points.

Cystine↗
PubMed2000

Role of disulfides and sulfhydryl groups in agonist and antagonist binding in serotonin1A receptors from bovine hippocampus.

I. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT1A receptors from bovine hippocampus. 2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity. 3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites. 4. In addition, ligand-binding studies in presence of GTP-gamma-S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT1A receptor and the G-protein. 5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT1A receptors.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed2004

Ferredoxin:thioredoxin Reductase: Disulfide Reduction Catalyzed via Novel Site-specific [4Fe-4S] Cluster Chemistry.

Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe-2S] ferredoxin as the electron donor and an active site comprising a [4Fe-4S] cluster and a redox-active disulfide. This review summarizes structural and spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) and a chemically modified form, termed NEM-FTR, which provides a stable analog of the one-electron reduced catalytic intermediate. Detailed spectroscopic characterization of FTR and NEM-FTR using absorption, EPR, electron-nuclear double resonance, variable-temperature magnetic circular dichroism, resonance Raman and Mössbauer spectroscopies indicate that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation of a [4Fe-4S](2+) cluster to yield a unique type of S= 1/2 [4Fe-4S](3+) cluster with two cysteine residues ligated at a specific Fe site. The results provide the basis for a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific [4Fe-4S] cluster chemistry. A similar mechanism is proposed for direct [4Fe-4S]-mediated cleavage of the CoM-S-S-CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.

Journal Article↗