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PubMed1996

Contribution of the intramolecular disulfide bridge to the folding stability of REIv, the variable domain of a human immunoglobulin kappa light chain.

BACKGROUND: Immunoglobulin domains contain about 100 amino acid residues folded into two beta-sheets and stabilized in a sandwich by a conserved central disulfide bridge. Whether antibodies actually require disulfide bonds for stability has long been a matter of debate. The contribution made by the central disulfide bridge to the overall folding stability of the immunoglobulin REIv, the variable domain of a human kappa light chain, was investigated by introducing stabilizing amino acid replacements followed by removal of the disulfide bridge via chemical reduction or genetic substitution of the cysteine residues. RESULTS: Nine REIv variants were constructed by methods of protein engineering that have folding stabilities elevated relative wild-type REIv by (up to) 16.0 kJ mol-1. Eight of these variants can be cooperatively refolded after unfolding and chemical reduction of the disulfide bridge-in contrast to wildtype REIv. The stabilizing effect of one of these residue replacements (T39K) was rationalized by determining the structure of the respective REIv variant at 1.7 A. The loss of folding stability caused by reduction of the intramolecular disulfide bond is on average 19 kJ mol-1. Removal of the disulfide bridge by genetic substitution of C23 for valine resulted in a stable immunoglobulin domain in the context of the stabilizing Y32H amino acid exchange; again, REIv-C23V/Y32H has 18 kJ mol-1 less folding stability than REIv-Y32H. The data are consistent with the notion that all variants studied have the same overall three-dimensional structure with the disulfide bridge opened or closed. CONCLUSIONS: A comparison of the magnitude of the stabilizing effect exerted by the disulfide bond and the length of the mainchain loop framed by it suggests lowering of the entropy of the unfolded state as the sole source of the effect. Disulfide bonds are not necessary for proper folding of immunoglobulin variable domains and can be removed, provided the loss of folding stability is at least partly compensated by stabilizing amino acid exchanges.

Disulfides↗
PubMed2006

Mapping of protein disulfide bonds using negative ion fragmentation with a broadband precursor selection.

Fast mapping of disulfide bonds in proteins containing multiple cysteine residues is often required in order to assess the integrity of the tertiary structure of proteins prone to degradation and misfolding or to detect distinct intermediate states generated in the course of oxidative folding. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures utilizes the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode. Cleavages of cysteine side chains result in efficient dissociation of disulfide bonds and produce characteristic signatures in the fragment ion mass spectra. While cleavages of other side chains result in insignificant loss of mass and full retention of the peptide ion charge, dissociation of external disulfide bonds results in physical separation of two peptides and, therefore, significant changes of both mass and charge of fragment ions relative to the precursor ion. This feature allows the fragment ions generated by dissociation of external disulfide bonds to be easily detected and identified even if multiple precursor ions are activated simultaneously. Such broadband selection of precursor ions for consecutive activation is achieved by lowering the dc/rf amplitude ratio in the first quadrupole filter of a hybrid quadrupole time-of-flight mass spectrometer. The feasibility of the new method is demonstrated by partial mapping of disulfide bridges within a 37-kDa protein containing 16 cysteine residues and complete disulfide mapping within a lysozyme (14.5 kDa) containing 8 cysteine residues. In addition to detecting peptide pairs connected by a single external disulfide, the new method is also shown to be capable of identifying peptides containing both external and internal disulfide bonds. The two major factors determining the efficiency of disulfide mapping using the new methodology are the effectiveness of proteolysis and the ability of the resulting proteolytic fragments to form multiply charged negative ions.

Amino Acid Sequence↗
PubMed1991

Identification of bull protamine disulfides.

We have identified the disulfide cross-links in bull protamine by titrating intact bull sperm with dithiothreitol (DTT) and following the modification of each cysteine residue with tritiated iodoacetate. The derivatization of each cysteine was monitored by a combination of HPLC, peptide mapping, and protein sequencing. Analyses of total free sulfhydryls show that all seven of the bull protamine cysteines are cross-linked as disulfides in mature sperm. The first disulfide is reduced at a DTT:protamine cysteine (DTT:Cys) ratio of 0.3 and the last at a ratio of 2.0. Intra- and intermolecular disulfides were identified by correlating the reduction of specific disulfides with the dissociation of protamine from DNA in partially reduced sperm and sperm treated with N,N'-ethylenedimaleimide, a bifunctional disulfide cross-linking agent. Three intermolecular and two intramolecular disulfides were identified. The results of these experiments demonstrate that the amino- and carboxy-terminal ends of the bull protamine molecule are folded inward toward the center of the molecule and are locked in place, each by a single intramolecular disulfide bridge. Three intermolecular disulfides cross-link neighboring protamine molecules around the DNA helix in such a manner that the protamines cannot be dissociated from DNA without first reducing the interprotamine disulfides.

Amino Acid Sequence↗
PubMed1989

Urea dependence of thiol-disulfide equilibria in thioredoxin: confirmation of the linkage relationship and a sensitive assay for structure.

Thioredoxin contains a single disulfide bond that can be reduced without perturbing significantly the structure of the enzyme. Upon reduction of the disulfide, protein stability decreases. We have experimentally tested the expected linkage relationship between disulfide bond formation and protein stability for thioredoxin. In order to do this, it is necessary to measure the equilibrium constant for disulfide bond formation in both the folded and unfolded states of the protein. Using glutathione as a reference species, we have measured the equilibrium constant for forming the disulfide bond (effective concentration) in thioredoxin as a function of urea concentration. As a control, we show that urea per se does not interfere with our measurements of thiol-disulfide equilibrium constants. Comparison of the values obtained for disulfide bond formation in the folded and unfolded states with the free energies for unfolding oxidized and reduced thioredoxin using circular dichroism confirms the expected linkage relationship. The urea dependence of thiol-disulfide equilibria provides a sensitive assay for folded structure in peptides or proteins. The method should also be useful to evaluate the stabilizing or destabilizing effect of natural or genetically engineered disulfides in proteins. In future work, the effects of amino acid substitutions on disulfide bond formation could be evaluated individually in the native and unfolded states of a protein.

Bacterial Proteins↗
PubMed1990

Beta subunit of (Na+ + K+)-ATPase contains three disulfide bonds.

Previous studies of titratable (Na+ + K+)-ATPase sulfhydryl groups have indicated the presence of one disulfide bond per mole of holoenzyme. This single disulfide cross-link was assigned to the beta subunit on the basis of the difference between the number of titrated "free" sulfhydryl groups and the total number of titrated sulfhydryl groups for each subunit [Esmann, M. (1982) Biochim. Biophys. Acta 688, 251; Kawamura, M., & Nagano, K. (1984) Biochim. Biophys. Acta 694, 27]. In the present study, beta-subunit tryptic peptides containing disulfide cross-links were identified and purified by HPLC. Two new peptides were generated from each disulfide-bonded peptide by reduction with dithiothreitol, and the amino acid compositions of these reduced peptides were determined. The data demonstrate that there are three disulfide bonds in the native beta subunit: 125Cys-148Cys, 158Cys-174Cys, and 212Cys-275Cys. The number of disulfide bonds in the beta subunit was also estimated by titration of sulfhydryl groups with [14C]iodoacetamide. Six sulfhydryl groups were identified: two sulfhydryl groups were titrated without prior reduction, and four were identified only after reduction of the protein with dithiothreitol. These data, suggesting that the beta subunit contains two disulfide bonds, are inconsistent with the peptide isolation experiments, which directly identified three disulfide bonds in the beta subunit. This inconsistency was resolved by demonstrating that approximately 20% of each disulfide bond in the beta subunit was reduced prior to the start of the experiment, resulting in an underestimation of the number of disulfide-bonded sulfhydryl groups in the beta subunit from the titration experiments.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed2006

Allosteric disulfide bonds.

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.

Allosteric Site↗
PubMed2000

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.

Animals↗
PubMed1998

Porcine submaxillary mucin forms disulfide-linked multimers through its amino-terminal D-domains.

COS-7 cells expressing 1,360 residues from the amino terminus of porcine submaxillary mucin were used to determine whether this region, containing the D1, D2, and D3 domains, is involved in forming mucin multimers. Analysis of the proteins immunoprecipitated from the medium of transfected cells by reducing SDS-gel electrophoresis showed a single N-glycosylated protein with no indication of proteolytically processed forms. Without prior reduction, only two proteins, corresponding to monomeric and disulfide-linked trimeric species, were observed. The expressed protein devoid of N-linked oligosaccharides also formed trimers, but was secreted from cells in significantly less amounts than glycosylated trimers. Pulse-chase studies showed that the disulfide-linked trimers were assembled inside the cells no earlier than 30 min after protein synthesis commenced and after the intracellular precursors were N-glycosylated. Trimer formation was inhibited in cells treated with brefeldin A, monensin, chloroquine, or bafilomycin A1, although only brefeldin A prevented the secretion of the protein. These results suggest that trimerization takes place in compartments of the Golgi complex in which the vacuolar H+-ATPase maintains an acidic pH. Coexpression in the same cells of the amino-terminal region and the disulfide-rich carboxyl-terminal domain of the mucin showed that these structures were not disulfide-linked with one another. Cells expressing a DNA construct encoding a fusion protein between the amino- and carboxyl-terminal regions of the mucin secreted disulfide-linked dimeric and high molecular weight multimeric species of the recombinant mucin. The presence of monensin in the medium was without effect on dimerization, but inhibited the formation of disulfide-linked multimers. These studies suggest that disulfide-linked dimers of mucin are subsequently assembled into disulfide-linked multimers by the amino-terminal regions. They also suggest that the porcine mucin forms branched disulfide-linked multimers. This ability of the amino-terminal region of mucin to aid in the assembly of multimers is consistent with its amino acid identities to the amino-terminal region of human von Willebrand factor, which also serves to form disulfide-linked multimers of this protein.

Animals↗
PubMed1999

Novel protein-disulfide isomerases from the early-diverging protist Giardia lamblia.

Protein-disulfide isomerase is essential for formation and reshuffling of disulfide bonds during nascent protein folding in the endoplasmic reticulum. The two thioredoxin-like active sites catalyze a variety of thiol-disulfide exchange reactions. We have characterized three novel protein-disulfide isomerases from the primitive eukaryote Giardia lamblia. Unlike other protein-disulfide isomerases, the giardial enzymes have only one active site. The active-site sequence motif in the giardial proteins (CGHC) is characteristic of eukaryotic protein-disulfide isomerases, and not other members of the thioredoxin superfamily that have one active site, such as thioredoxin and Dsb proteins from Gram-negative bacteria. The three giardial proteins have very different amino acid sequences and molecular masses (26, 50, and 13 kDa). All three enzymes were capable of rearranging disulfide bonds, and giardial protein-disulfide isomerase-2 also displayed oxidant and reductant activities. Surprisingly, the three giardial proteins also had Ca(2+)-dependent transglutaminase activity. This is the first report of protein-disulfide isomerases with a single active site that have diverse roles in protein cross-linking. This study may provide clues to the evolution of key functions of the endoplasmic reticulum in eukaryotic cells, protein disulfide formation, and isomerization.

Amino Acid Sequence↗
PubMed2001

Relative roles of albumin and ceruloplasmin in the formation of homocystine, homocysteine-cysteine-mixed disulfide, and cystine in circulation.

Disulfide forms of homocysteine account for >98% of total homocysteine in plasma from healthy individuals. We recently reported that homocysteine reacts with albumin-Cys(34)-S-S-cysteine to form homocysteine-cysteine mixed disulfide and albumin-Cys(34) thiolate anion. The latter then reacts with homocystine or homocysteine-cysteine mixed disulfide to form albumin-bound homocysteine (Sengupta, S., Chen, H., Togawa, T., DiBello, P. M., Majors, A. K., Büdy, B., Ketterer, M. E., and Jacobsen, D. W. (2001) J. Biol. Chem. 276, 30111-30117). We now extend these studies to show that human albumin, but not ceruloplasmin, mediates the conversion of homocysteine to its low molecular weight disulfide forms (homocystine and homocysteine-cysteine mixed disulfide) by thiol/disulfide exchange reactions. Only a small fraction of homocystine is formed by an oxidative process in which copper bound to albumin, but not ceruloplasmin, mediates the reaction. When copper is removed from albumin by chelation, the overall conversion of homocysteine to its disulfide forms is reduced by only 20%. Ceruloplasmin was an ineffective catalyst of homocysteine oxidation, and immunoprecipitation of ceruloplasmin from human plasma did not inhibit the capacity of plasma to mediate the conversion of homocysteine to its disulfide forms. In contrast, ceruloplasmin was a highly efficient catalyst for the oxidation of cysteine and cysteinylglycine to cystine and bis(-S-cysteinylglycine), respectively. However, when thiols (cysteine and homocysteine) that are disulfide-bonded to albumin-Cys(34) are removed by treatment with dithiothreitol to form albumin-Cys(34)-SH (mercaptalbumin), the conversion of homocysteine to its disulfide forms is completely blocked. In conclusion, albumin mediates the formation of disulfide forms of homocysteine by thiol/disulfide exchange, whereas ceruloplasmin converts cysteine to cystine by copper-dependent autooxidation.

Ceruloplasmin↗
PubMed2003

MODIP revisited: re-evaluation and refinement of an automated procedure for modeling of disulfide bonds in proteins.

There have been several attempts to stabilize proteins through the introduction of engineered disulfide bonds. For reasons that are currently unclear, these have met with mixed success. Hence identification of locations where introduction of a disulfide cross-link will lead to protein stabilization is still a challenging task. A computational procedure, MODIP, was introduced more than a decade ago to select sites in protein structures that have the correct geometry for disulfide formation when replaced by Cys. In this study, we re-evaluated the stereochemical criteria used by MODIP for the selection and gradation of sites for modeling disulfides. We introduced steric criteria to check for energetically unfavorable non-bonded contacts with the modeled disulfide, since these can considerably offset the stabilizing effect of the cross-link. The performance of the refined procedure was checked for its ability to correctly predict naturally occurring disulfide bonds in proteins. A set of proteins in which disulfide bonds were introduced experimentally were analyzed with respect to MODIP predictions, stability and other parameters such as accessibility, residue depth, B-factors of the mutated sites, change in volume upon mutation and loop length enclosed by the disulfide. The analysis suggests that in addition to proper stereochemistry, stabilizing disulfides occur in regions of low depth, relatively high mobility, have a loop length greater than 25 and where the disulfide typically occupies a volume less than or equal to that of the original residues.

Computational Biology↗
PubMed1999

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.

Amino Acid Substitution↗
PubMed2003

Homo-oligomerization of the porcine reproductive and respiratory syndrome virus nucleocapsid protein and the role of disulfide linkages.

As a step toward understanding the assembly pathway of the porcine reproductive and respiratory syndrome virus (PRRSV), the oligomeric properties of the nucleocapsid (N) protein were investigated. In this study, we have demonstrated that under nonreducing conditions the N protein forms disulfide-linked homodimers. However, inclusion of an alkylating agent (N-ethylmaleimide [NEM]) prevented disulfide bond formation, suggesting that these intermolecular disulfide linkages were formed as a result of spurious oxidation during cell lysis. In contrast, N protein homodimers isolated from extracellular virions were shown to have formed NEM-resistant intermolecular disulfide linkages, the function of which is probably to impart stability to the virion. Pulse-chase analysis revealed that N protein homodimers become specifically disulfide linked within the virus-infected cell, albeit at the later stages of infection, conceivably when the virus particle buds into the oxidizing environment of the endoplasmic reticulum. Moreover, NEM-resistant disulfide linkages were shown to occur only during productive PRRSV infection, since expression of recombinant N protein did not result in the formation of NEM-resistant disulfide-linked homodimers. Mutational analysis indicated that of the three conserved cysteine residues in the N protein, only the cysteine at position 23 was involved in the formation of disulfide linkages. The N protein dimer was shown to be stable both in the presence and absence of intermolecular disulfide linkages, indicating that noncovalent interactions also play a role in dimerization. Non-disulfide-mediated N protein interactions were subsequently demonstrated both in vitro by the glutathione S-transferase (GST) pull-down assay and in vivo by the mammalian two-hybrid assay. Using a series of N protein deletion mutants fused to GST, amino acids 30 to 37 were shown to be essential for N-N interactions. Furthermore, since RNase A treatment markedly decreased N protein-binding affinity, it appears that at least in vitro, RNA may be involved in bridging N-N interactions. In cross-linking experiments, the N protein was shown to assemble into higher-order structures, including dimers, trimers, tetramers, and pentamers. Together, these findings demonstrate that the N protein possesses self-associative properties, and these likely provide the basis for PRRSV nucleocapsid assembly.

Animals↗
PubMed2005

Effects of disulfide bonds on folding behavior and mechanism of the beta-sheet protein tendamistat.

Tendamistat, a small disulfide-bonded beta-sheet protein, and its three single/double-disulfide mutants are investigated by using a modified Gō-like model, aiming to understand the folding mechanism of disulfide-bonded protein as well as the effects of removal of disulfide bond on the folding process. Our simulations show that tendamistat and its two single-disulfide mutants are all two-state folders, consistent with the experimental observations. It is found that the disulfide bonds as well as three hydrogen bonds between the N-terminal loop-0 and strand-6 are of significant importance for the folding of tendamistat. Without these interactions, their two-state behaviors become unstable and the predictions of the model are inconsistent with experiments. In addition, the effect of disulfide bonds on the folding process are studied by comparing the wild-type tendamistat and its two mutants; it is found that the removal of either of the C11-C27 or C45-C73 disulfide bond leads to a large decrease in the thermodynamical stability and loss of structure in the unfolded state, and the effect of the former is stronger than that of the later. These simulation results are in good agreement with experiments and, thus, validate our model. Based on the same model, the detailed folding pathways of the wild-type tendamistat and two mutants are studied, and the effect of disulfide bonds on the folding kinetics are discussed. The obtained results provide a detailed folding picture of these proteins and complement experimental findings. Finally, the folding nuclei predicted to be existent in this protein tendamistat as well as its mutants are firstly identified in this work. The positions of the nucleus are consistent with those argued in experimental studies. Therefore, a nucleation/growth folding mechanism that can explain the two-state folding manner is clearly characterized. Moreover, the effect by the removal of each disulfide bond on the folding thermodynamics and dynamics can also be well interpreted from their influence on the folding nucleus. The implementation of this work indicates that the modified Gō-like model really describes the folding behavior of protein tendamistat and could be used to study the folding of other disulfide-bonded proteins.

Biophysical Phenomena↗
PubMed1999

Parallel syntheses of disulfide inhibitors of the thioredoxin redox system as potential antitumor agents.

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.

Animals↗
PubMed1992

Domain-dependent protein folding is indicated by the intracellular kinetics of disulfide bond formation of human chorionic gonadotropin beta subunit.

We have measured the intracellular rates of formation of the six disulfide bonds in the human chorionic gonadotropin beta subunit (hCG-beta) to determine whether the folding pathway of this molecule can be described by a simple sequential model. If such a model is correct, the formation of disulfide bonds, which is indicative of tertiary structural changes during protein folding, should occur in a discrete order. The individual rates of disulfide bridging were determined by identifying the extent of disulfide bond formation in hCG-beta intermediates purified from choriocarcinoma cells that had been metabolically labeled for 40 to 120 s and chased for 0 to 25 min. The results of these kinetic studies describe a folding pathway in which the disulfide bonds between cysteines 34-88, 38-57, 9-90 and 23-72 stabilize, in a discrete order, the putative domain(s) involving amino acids 1-90 of hCG-beta. However, the S-S bonds 93-100 and 26-110 begin to form before the complete formation of the disulfide bonds that stabilize the amino acid 1-90 domain(s), and continue to form after complete formation of these disulfide bonds, suggesting that hCG-beta does not fold by a simple sequential pathway. The order of completion of each of the six disulfide bonds of hCG-beta is: 34-88 (t1/2 = 1-2 min), 38-57 (t1/2 = 2-3 min), 9-90 and 23-72, 93-100, and 26-110. Moreover, 60-100% of each of the six disulfide bonds form posttranslationally, and nonnative disulfide bonds do not form in detectable amounts during intracellular folding of hCG-beta.

Choriocarcinoma↗
PubMed1994

Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

Animals↗
PubMed1998

Inhibition of the delayed rectifier K current in guinea-pig cardiomyocytes by thiamine tetrahydrofurfuryl disulfide.

We examined effect of thiamine tetrahydrofurfuryl disulfide on electrophysiological characteristics of single atrial myocytes, obtained by digestion of guinea-pig heart, using collagenase. Membrane potential and ion channel current in the atrial myocytes were recorded by the patch clamp method. Thiamine tetrahydrofurfuryl disulfide prolonged action potentials at cycle lengths from 250 to 10,000 ms. The degree of thiamine tetrahydrofurfuryl disulfide-induced prolongation was similar among these cycle lengths. Thiamine tetrahydrofurfuryl disulfide inhibited the delayed rectifier K+ current, without affecting Ca2+ current and inward-rectifier K+ current. Thiamine tetrahydrofurfuryl disulfide blocked the delayed rectifier K+ current in voltage- and time-independent manner, indicating that thiamine tetrahydrofurfuryl disulfide blocked both subtypes of the delayed rectifier K+ current (rapid and slow components). Thiamine, the parent molecule of thiamine tetrahydrofurfuryl disulfide, blocked the delayed rectifier K+ current only when thiamine was applied intracellularly. Thiamine tetrahydrofurfuryl disulfide may be converted to thiamine in the cytoplasm, and then may block the the delayed rectifier K+ channel from the intracellular side. Although thiamine tetrahydrofurfuryl disulfide (or thiamine) has some of the properties of class III antiarrhythmics agents, thiamine tetrahydrofurfuryl disulfide did not exhibit reverse use-dependent prolongation of action potential.

Action Potentials↗