Formation Of Disulphide Bridges Research Articles

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.

Source of tryptone in growth medium affects oxidative stress resistance in Escherichia coli.

To investigate the influence of the source of tryptone in the growth medium on the resistance of Escherichia coli to various types of oxidative stress. Cultures of Escherichia coli MG1655 were grown in Luria-Bertani (LB) medium at 37 degrees C to stationary phase, harvested, and subsequently subjected to various types of oxidative stress. A marked difference in oxidative stress sensitivity was observed depending on the origin of the tryptone in the LB medium used to grow the cultures. Cells harvested from LB containing tryptone from source x (LBx) were more sensitive to inactivation by the superoxide generating compound plumbagin and by t-butyl peroxide, and to growth inhibition by the lactoperoxidase enzyme system, than cells harvested from LB containing tryptone from source y (LBy). By monitoring expression of a panel of stress gene promotors linked to the gfp (green fluorescent protein) gene, and using Delta2-22 alkaline phosphatase as a probe for disulphide bridge formation from protein sulphydryl groups, it was demonstrated that a greater cytoplasmic oxidative stress existed in cells during growth in LBy than in LBx. Depending on the source of tryptone, bacteria may experience different levels of oxidative stress in tryptone-containing nonselective growth media. Although these levels of oxidative stress are subinhibitory, they may trigger a stress response that makes the bacteria more resistant to a subsequent exposure to a lethal or inhibitory level of oxidative stress. This work highlights the importance of controlling very subtle differences in composition of nonselective growth media in studies on bacterial physiology.

Ab initio modelling of the N-terminal domain of the secretin receptors

G protein coupled receptors of the secretin family are activated by peptide hormones of about 30 residues in length. There is considerable sequence homology within both the hormone and receptor families. The receptors possess in addition to the integral membrane domain a characteristic extracellular domain of about 120 residues in length, having conserved cysteine residues, which are involved in disulphide bridge formation, and tryptophanes, which have been shown to be critical for hormone binding. This extracellular domain does not have detectable homology to any known protein fold. In order to be able to propose a structure for this domain we have used ab initio prediction methods combined with constraints based on experimental results for the disulphide connectivity. The results of computational tools for predicting secondary structure and accessibility, together with ligand binding and mutational data and other structural considerations were used in the ab initio protein folding programs dragon and gadget and also the simpler program ramble, which was able to explore different permutations of disulphide bond connectivity, tryptophan side chain orientation and chain topology. The methods generated a limited number of plausible models but no single unique solution was found under the constraints. One of these was refined into a full atomic model that contained a possible peptide binding site comprising the most conserved residues.

Formation of nuclear matrix filaments by p27 BBP/eIF6

p27 BBP/eIF6 is an evolutionarily conserved protein necessary for ribosome biogenesis which was cloned in mammals for its ability to bind the cytodomain of β4 integrin. In cultured cells, a conspicuous fraction of p27 BBP/eIF6 is associated with the intermediate filaments/nuclear matrix (IF/NM) cytoskeleton. The mechanism of this association is not known. Here we show that in epidermis p27 BBP/eIF6 is naturally associated with IF/NM. To analyze the intrinsic capability of p27 BBP/eIF6 to generate cytoskeletal networks, the properties of the pure, recombinant, untagged protein were studied. Recombinant p27 BBP/eIF6 binds β4 integrin. Upon dialysis against IF buffer, p27 BBP/eIF6 forms polymers which, strikingly, have a morphology identical to NM filaments. Cross-linking experiments suggested that polymerization is favored by the formation of disulphide bridges. These data suggest that p27 BBP/eIF6 is associated with the cytoskeleton, and contributes to formation of NM filaments. These findings help to settle the controversy on nuclear matrix.

Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro.

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K(+) channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80 h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the alpha-helix within 5 h, followed by the formation of the beta-sheet within 22 h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the alpha-helix and two- or three-disulphide-bridged sMTX for the beta-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K(+) currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

Evolution of maurotoxin conformation and blocking efficacy towards Shaker B channels during the course of folding and oxidation in vitro

Maurotoxin (MTX) is a 34-mer scorpion toxin cross-linked by four disulphide bridges that acts on various K+ channels, including the voltage-gated Shaker B subtype. In the present study, we have investigated over 80h: (1) the time-course of folding of synthetic MTX (sMTX) by CD analysis; (2) the kinetics of disulphide bridge formation by MS; and (3) the potency of MTX in blocking Shaker B currents during the combined process of its in vitro folding and oxidation. From the CD data, we show that stable secondary structures of sMTX evolve sequentially over time, with the appearance of the α-helix within 5h, followed by the formation of the β-sheet within 22h. Using MS analysis, the sMTX intermediates were also found to appear sequentially from the least (one-disulphide-bridged sMTX) to the most oxidized species (native-like, four-disulphide-bridged sMTX). The time course of formation of secondary structures coincides mainly with the occurrence of one-disulphide-bridged sMTX for the α-helix and two- or three-disulphide-bridged sMTX for the β-sheet. On-line electrophysiological recordings, which measure sMTX blocking efficacy on K+ currents during its folding and oxidation, were performed on Shaker B channels expressed in Xenopus oocytes. Unexpectedly, the results demonstrate that sMTX is highly potent at the initial stage of oxidation, whereas its blocking activity can be transiently and dramatically reduced at later stages during the course of folding/oxidation before it reaches full bioactivity. These data suggest that formation of disulphide bridges can both physically stabilize and alter the bioactive three-dimensional structure of sMTX.

Engineering new peptidic inhibitors from a natural chymotrypsin inhibitor.

Three model peptides of different sizes (17-24 amino acid residues) mimicking the chymotrypsin inhibitor SCGI (a peptide of 35 amino acid residues) isolated from Schistocerca gregaria were designed and prepared by convergent peptide synthesis. Selective formation of disulphide bridges in the closing step was achieved without selective protection of cysteine residues. The natural pattern of the two disulphide bridges was determined by 2D homonuclear 1H NMR techniques. All three model peptides were characterized by amino acid analysis. MS and CD spectra. Preliminary results revealed that the two smaller model peptides exhibit no Inhibitory activity, whereas the larger one shows limited inhibition of chymotrypsin.

Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms

Spinach starch debranching enzyme, a limit dextrinase or pullulanase (EC 3.2.1.41), is a monomeric protein of 100 kDa that produces up to seven coexisting and mutually interconvertible isomers of different specific activity, a phenomenon that has been termed microheterogeneity and for which a structural explanation has not yet been presented. The enzyme can be activated by reduction, in particular by thiol reagents, and inactivated by oxidation and the concomitant change of the patterns of its isomeric forms could be quantified by chromatofocusing. The hypothesis was examined that reduction of the enzyme’s thiol groups shifts the isomer pattern towards the forms with a higher specific activity while oxidation favours the less active forms. Using TCEP as reductant only the form with the highest specific activity was obtained. This form was almost inaccessible for proteolysis by trypsin while the oxidized and GSH-activated enzyme yielded four peptides when treated with trypsin. Their sequence indicated cleavage predominantly of loops connecting the β-strands and α-helices of the (β/α) 8-barrel which forms the catalytic site of the pullulanase. Formation of various disulphide bridges between the loops connecting the barrel structures – predominantly on one side – may be the reason for the microheterogeneity of the spinach pullulanase. In vivo, the enzyme maintains its activated state due to the high concentration of GSH in the chloroplast. However, the chloroplast’s pH shifts from day (pH 8) to night (pH 7) and thus could also alter the activity of the protein in accordance with the required function in starch metabolism.

Structural Evidence for a Possible Role of Reversible Disulphide Bridge Formation in the Elasticity of the Muscle Protein Titin

Background: The giant muscle protein titin contributes to the filament system in skeletal and cardiac muscle cells by connecting the Z disk and the central M line of the sarcomere. One of the physiological functions of titin is to act as a passive spring in the sarcomere, which is achieved by the elastic properties of its central I band region. Titin contains about 300 domains of which more than half are folded as immunoglobulin-like (Ig) domains. Ig domain segments of the I band of titin have been extensively used as templates to investigate the molecular basis of protein elasticity. Results: The structure of the Ig domain I1 from the I band of titin has been determined to 2.1 Å resolution. It reveals a novel, reversible disulphide bridge, which is neither required for correct folding nor changes the chemical stability of I1, but it is predicted to contribute mechanically to the elastic properties of titin in active sarcomeres. From the 92 Ig domains in the longest isoform of titin, at least 40 domains have a potential for disulphide bridge formation. Conclusions: We propose a model where the formation of disulphide bridges under oxidative stress conditions could regulate the elasticity of the I band in titin by increasing sarcomeric resistance. In this model, the formation of the disulphide bridge could refrain a possible directed motion of the two β sheets or other mechanically stable entities of the I1 Ig domain with respect to each other when exposed to mechanical forces.

Formation of disulphide bonds during secretion of proteins through the periplasmic-independent type I pathway.

In this work, we have investigated whether the bacterial type I secretion pathway, which does not have a periplasmic intermediate of the secreted protein, allows the formation of disulphide bridges. To this end, the formation of disulphide bonds has been studied in an antibody single-chain Fv (scFv) fragment secreted by the Escherichia coli haemolysin (Hly) transporter (a paradigm of type I secretion). The scFv antibody fragment was used as a disulphide bond and protein-folding reporter, as it contains two disulphide bridges that are required for its correct folding (i.e. to preserve its antigen-binding activity). We show that an scFv-HlyA hybrid secreted by Hly type I transporter (TolC, HlyB, HlyD) is accumulated in the extracellular medium with the disulphide bonds correctly formed. Neither periplasmic and inner membrane-bound Dsb enzymes (e.g. DsbC, DsbG, DsbB and DsbD) nor cytoplasmic thioredoxins (TrxA and TrxC) were required for scFv-HlyA oxidation. However, a mutation of the thioredoxin reductase gene (trxB), which leads to the cytoplasmic accumulation of the oxidized forms of thioredoxins, had a specific inhibitory effect on the Hly-dependent secretion of disulphide-containing proteins. These data suggest that premature cytoplasmic oxidation of the substrate may interfere with the secretion process. Taken together, these results indicate not only that the type I system tolerates secretion of disulphide-containing proteins, but also that disulphide bonds are specifically formed during the passage of the polypeptide through the export conduit.

Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

BackgroundCa2+-ATPases of endoplasmic reticulum (SERCAs) are responsible for maintenance of the micro- to millimolar Ca2+ ion concentrations within the endoplasmic reticulum (ER) of eukaryotic cells. This intralumenal Ca2+ storage is important for the generation of Ca2+ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca2+ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity.ResultsWe show here that in PC12 cells, depletion of ER Ca2+ by EGTA, as well as inhibition of disulphide bridge formation within the ER by dithiotreitol or inhibition of N-glycosylation by tunicamycin, led to a 2- to 3-fold increase of the SERCA-mediated 45Ca2+ transport to microsomes isolated from cells exposed to these stress agents. The time course of this response corresponded to that for transcriptional upregulation of ER stress proteins, as well as to the increase in the SERCA2b mRNA, as we recently observed in an independent study.ConclusionsThese findings provide the first functional evidence for the increase of SERCA pumping capacity in cells subjected to the ER stress. Since at least three different and unrelated mechanisms of eliciting the ER stress response were found to cause this functional upregulation of Ca2+ transport into the ER, these results support the existence of a coupling between the induction of the UPR pathway in general, and the regulation of expression of at least one of the SERCA pump isoforms.

Effect of mobile phase on the oligomerization state of α-helical coiled-coil peptides during high-performance size-exclusion chromatography

Important structural motifs involving amphipathic helices include two-stranded and multiple-stranded coiled-coils. High-performance size-exclusion chromatography (HPSEC) is a useful tool to examine both the oligomerization state of coiled-coils as well as the stability of such motifs, due to the facile manipulation of the mobile phase and the lack of interaction of the peptide solutes with the stationary phase. In the present study, HPSEC was applied to two series of de novo designed model amphipathic α-helical peptides with the sequences (1) Ac–(E–A–L–K–A–E–I) n –E–A–C–K–A–amide, where n=1 or 3, Ac–E–I–(E–A–L–K–A–E–I) 4–E–A–C–K–A–amide and (2) Ac–(K–L–E–A–L–E–A) n –amide, where n=1, 2 or 4. Observation of the retention behaviour of Series 1 under both denaturing and non-denaturing conditions at pH 7.0 offered insights into the effect of polypeptide chain length and disulphide bridge formation on the stability of α-helical coiled-coils. In contrast, the Series 2 peptides showed promise as peptide standards to monitor the effect of environment on the multi-strandedness of coiled-coils, since the 28-residue peptide of this series was eluted as a monomer, dimer or trimer depending on mobile phase conditions.

The solubilization of the basic subunit of sugarbeet seed 11-S globulin during priming and early germination

AbstractThe basic B-subunit of the seed storage protein 11-S globulin (an 11-S-legumin type protein) is the major polypeptide in soluble protein extracts from primed sugarbeet (Beta vulgarisL.) seeds. In contrast, only a small amount of this protein is present in corresponding extracts from untreated dry mature seeds. Here, and as for all 11-S globulins described so far, the B-chain is linked to other polypeptide(s), corresponding most presumably to an acidic A-chain, through the formation of disulphide bridge(s). Polyacrylamide gel electrophoretic analyses of total and soluble protein extracts from untreated and primed seeds strongly indicate that this priming-induced solubilization of the B-chain resulted from an endoproteolytic attack on the A-chain. Microscopical immunolocalization showed a uniform distribution of the 11-S globulin B-chain over the protein bodies of the embryonic cells from the untreated seeds. For the primed seeds, however, the B-subunit of 11-S globulin diffused out of the protein bodies and invaded the cytosolic compartment. This phenomenon occurred independently of the manner of priming, being observed with hydroprimed and osmoprimed seeds, as well as with sugarbeet seeds that had been primed by a prehydration treatment. Quantitative analyses of the amounts of soluble 11-S globulin B-chain have enabled the priming of sugarbeet seeds to be optimized.

Oxygen Binding and Aggregation of Hemoglobin from the Common European Frog, Rana temporaria

The hemoglobin from the European frog, Rana temporaria, consists of one major and three minor components. The tetramers aggregate upon deoxygenation notably at pH 7.3. Aggregation due to formation of disulphide bridges, as occurs in related species, was observed only in polyacrylamide gels. The hemolysate showed a pronounced Bohr effect. Oxygen affinity decreased with increasing hemoglobin concentration, indicating that aggregation affects the functional properties of the hemolysate. Oxygen binding equilibria of unfractionated hemolysate are insensitive to chloride and show low sensitivity to ATP. Analysis of oxygen equilibrium curves in terms of the two-state allosteric model (MWC) showed that pH change exerted a greater effect on the association constant of the oxygenated state (KR) than that of the deoxy state (KT). The number of interacting binding sites (q) increased with hemoglobin concentration. Cooperativity of oxygen binding, evaluated as Hill coefficient n, never exceeded the value of 3.0. Earlier studies on hemoglobin and blood from this and related species, report significantly higher n values at high O2 saturation. Molecular adaptive aspects are discussed.

Separation of a bioactive cyclic peptide and its oligomeric forms by micellar electrokinetic chromatography

The monomer and the oligomers of a bioactive cyclic peptide were separated using micellar electrokinetic chromatography (MEKC) in order to determine if oligomeric forms of the peptide were present or not. Producing cyclic peptides involves the formation of an intramolecular disulphide bridge. During cyclisation, intermolecular disulphide bonds may be formed, thus resulting in unwanted oligomeric forms of the peptide. The cyclic peptide studied was shown by size-exclusion chromatography to be capable of forming a monomer, dimer, trimer and tetramer. The higher resolution MEKC provides was needed to separate further components of the oligomers. In phosphate buffer pH 7.5 containing sodium dodecyl sulphate 20 species could be separated in 18 min. Both linear and cyclic oligomeric isomers appeared to be present.

NMR structure of inactivation gates from mammalian voltage-dependent potassium channels.

The electrical signalling properties of neurons originate largely from the gating properties of their ion channels. N-type inactivation of voltage-gated potassium (Kv) channels is the best-understood gating transition in ion channels, and occurs by a 'ball-and-chain' type mechanism. In this mechanism an N-terminal domain (inactivation gate), which is tethered to the cytoplasmic side of the channel protein by a protease-cleavable chain, binds to its receptor at the inner vestibule of the channel, thereby physically blocking the pore. Even when synthesized as a peptide, ball domains restore inactivation in Kv channels whose inactivation domains have been deleted. Using high-resolution nuclear magnetic resonance (NMR) spectroscopy, we analysed the three-dimensional structure of the ball peptides from two rapidly inactivating mammalian K. channels (Raw3 (Kv3.4) and RCK4 (Kv1.4)). The inactivation peptide of Raw3 (Raw3-IP) has a compact structure that exposes two phosphorylation sites and allows the formation of an intramolecular disulphide bridge between two spatially close cysteine residues. Raw3-IP exhibits a characteristic surface charge pattern with a positively charged, a hydrophobic, and a negatively charged region. The RCK4 inactivation peptide (RCK4-IP) shows a similar spatial distribution of charged and uncharged regions, but is more flexible and less ordered in its amino-terminal part.

The role of the novel disulphide ring in the active site of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens.

All cysteines in methanol dehydrogenase (MDH) from Methylobacterium extorquens are involved in intra-subunit disulphide bridge formation. One of these is between adjacent cysteine residues which form a novel ring structure in the active site. It is readily reduced, the reduced enzyme being inactive in electron transfer to cytochrome cL. The inactivation is not a result of major structural change or to modification of the prosthetic group pyrrolo-quinoline quinone (PQQ). The reduced enzyme appears to remain active with the artificial electron acceptor phenazine ethosulphate but this is because the dye re-oxidizes the adjacent thiols back to the original disulphide bridge. No free thiols were detected during the reaction cycle with cytochrome cL. Carboxymethylation of the thiols produced by reduction of the novel disulphide ring led to formation of active enzyme. Reconstitution of inactive Ca(2+)-free MDH with Ca2+ led to active enzyme containing the oxidized bridge and reduced quinol, PQQH2, consistent with the conclusion that no hydrogen transfer occurs between these groups in the active site. It is concluded that the disulphide ring in the active site of MDH does not function as a redox component of the reaction. The disulphide ring has no special function in the process of Ca2+ incorporation into the active site. It is suggested that this novel structure might function in the stabilization or protection of the free radical semiquinone form of the prosthetic group (PQQH.) from solvent at the entrance to the active site.

A trout growth hormone is expressed, correctly folded and partially glycosylated in the leaves but not the seeds of transgenic plants

The growth hormone gene of the rainbow trout (tGH-II) was expressed in leaves of transgenic tobacco plants and seeds of transgenicArabidopsis plants using tissue-specific promoters. Although in the leaves and the seeds comparble amounts of tGH-II mRNA could be detected, the protein could only be identified in the tobacco leaves. Passage of the hormone into the secretory pathway, mediated by the signal sequence of the extracellular tobacco PRI-b (pathogenesis-related) protein, resulted in correct disulphide bridge formation and (partial) glycosylation of the hormone. In contrast, cytoplasmic expression resulted in misfolding and partial breakdown of the protein. The data demonstrate that synthesis, folding and glycosylation of heterologous proteins in plants is dependent both on subcellular location as well as on the tissue or cell type in which the protein is expressed.

Clinical implications of hepatitis B virus envelope protein variation.

The envelope proteins of hepatitis B virus are highly conserved. They are involved in viral attachment to hepatocytes, virion assembly and secretion and induction of humoral and T-cell immune responses. The major neutralisation epitope is located within the S protein and is conformation dependent, probably due to the formation of disulphide bridges. Variation in the envelope proteins of hepatitis B virus has been described in vaccinees and in patients receiving monoclonal and polyclonal antibody therapy. These arise through point mutations in the surface gene and appear to be selected by the immune response. The emergence of such variants has clinical relevance, in particular with regard to vaccine failure and diagnostic difficulties.

The structure of secreted mucins isolated from the adherent mucus gel: comparison with the gene products.

The structure of secreted mucins isolated from the adherent mucus gel: comparison with the gene products.