The Experts below are selected from a list of 552 Experts worldwide ranked by ideXlab platform
Jon Beckwith - One of the best experts on this subject based on the ideXlab platform.
-
bacterial species exhibit diversity in their mechanisms and capacity for protein disulfide bond formation
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Rachel J Dutton, Dana Boyd, Mehmet Berkmen, Jon BeckwithAbstract:Protein disulfide bond formation contributes to the folding and activity of many exported proteins in bacteria. However, information about disulfide bond formation is limited to only a few bacterial species. We used a multifaceted bioinformatic approach to assess the capacity for disulfide bond formation across this biologically diverse group of organisms. We combined data from a cysteine counting method, in which a significant bias for even numbers of cysteine in proteins is taken as an indicator of disulfide bond formation, with data on the presence of homologs of known disulfide bond formation enzymes. These combined data enabled us to make predictions about disulfide bond formation in the cell envelope across bacterial species. Our bioinformatic and experimental results suggest that many bacteria may not generally oxidatively fold proteins, and implicate the bacterial homolog of the enzyme vitamin K epoxide reductase, a protein required for blood clotting in humans, as part of a disulfide bond formation pathway present in several major bacterial phyla.
-
the nonconsecutive disulfide bond of escherichia coli phytase appa renders it dependent on the protein disulfide isomerase dsbc
Journal of Biological Chemistry, 2005Co-Authors: Mehmet Berkmen, Dana Boyd, Jon BeckwithAbstract:The formation of protein disulfide bonds in the Escherichia coli periplasm by the enzyme DsbA is an inaccurate process. Many eukaryotic proteins with nonconsecutive disulfide bonds expressed in E. coli require an additional protein for proper folding, the disulfide bond isomerase DsbC. Here we report studies on a native E. coli periplasmic acid phosphatase, phytase (AppA), which contains three consecutive and one nonconsecutive disulfide bonds. We show that AppA requires DsbC for its folding. However, the activity of an AppA mutant lacking its nonconsecutive disulfide bond is DsbC-independent. An AppA homolog, Agp, a periplasmic acid phosphatase with similar structure, lacks the nonconsecutive disulfide bond but has the three consecutive disulfide bonds found in AppA. The consecutively disulfide-bonded Agp is not dependent on DsbC but is rendered dependent by engineering into it the conserved nonconsecutive disulfide bond of AppA. Taken together, these results provide support for the proposal that proteins with nonconsecutive disulfide bonds require DsbC for full activity and that disulfide bonds are formed predominantly during translocation across the cytoplasmic membrane.
-
the role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the escherichia coli cytoplasm
Journal of Biological Chemistry, 1997Co-Authors: William A Prinz, Arne Holmgren, Fredrik Aslund, Jon BeckwithAbstract:In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.
-
the role of the thioredoxin and glutaredoxin pathways in reducing protein disulfide bonds in the escherichia colicytoplasm
Journal of Biological Chemistry, 1997Co-Authors: William A Prinz, Arne Holmgren, Fredrik Aslund, Jon BeckwithAbstract:In Escherichia coli, two pathways use NADPH to reduce disulfide bonds that form in some cytoplasmic enzymes during catalysis: the thioredoxin system, which consists of thioredoxin reductase and thioredoxin, and the glutaredoxin system, composed of glutathione reductase, glutathione, and three glutaredoxins. These systems may also reduce disulfide bonds which form spontaneously in cytoplasmic proteins when E. coli is grown aerobically. We have investigated the role of both systems in determining the thiol-disulfide balance in the cytoplasm by determining the ability of protein disulfide bonds to form in mutants missing components of these systems. We find that both the thioredoxin and glutaredoxin systems contribute to reducing disulfide bonds in cytoplasmic proteins. In addition, these systems can partially substitute for each otherin vivo since double mutants missing parts of both systems generally allow substantially more disulfide bond formation than mutants missing components of just one system. Some of these double mutants were found to require the addition of a disulfide reductant to the medium to grow well aerobically. Thus, E. coli requires either a functional thioredoxin or glutaredoxin system to reduce disulfide bonds which appear after each catalytic cycle in the essential enzyme ribonucleotide reductase and perhaps to reduce non-native disulfide bonds in cytoplasmic proteins. Our results suggest the existence of a novel thioredoxin in E. coli.
-
identification of a protein required for disulfide bond formation in vivo
Cell, 1991Co-Authors: James C.a. Bardwell, Karen Mcgovern, Jon BeckwithAbstract:Abstract We describe a mutation ( dsbA ) that renders Escherichia coli severely defective in disulfide bond formation. In dsbA mutant cells, pulse-labeled β-lactamase, alkaline phosphatase, and OmpA are secreted but largely lack disulfide bonds. These disulfideless proteins may represent in vivo folding intermediates, since they are protease sensitive and chase slowly into stable oxidized forms. The dsbA gene codes for a 21,000 M r periplasmic protein containing the sequence cys-pro-his-cys, which resembles the active sites of certain disulfide oxidoreductases. The purified DsbA protein is capable of reducing the disulfide bonds of insulin, an activity that it shares with these disulfide oxidoreductases. Our results suggest that disulfide bond formation is facilitated by DsbA in vivo.
Urs Jans - One of the best experts on this subject based on the ideXlab platform.
-
stereoisomer specific reaction of hexabromocyclododecane with reduced sulfur species in aqueous solutions
Chemosphere, 2019Co-Authors: Xianmiao Zhang, Urs Jans, John H Wilson, Ariel J Lawson, Edward G HohensteinAbstract:Abstract The individual degradation rates of the three dominant stereoisomers (α, β, γ) of hexabromocyclododecane (HBCDD) with Bisulfide and polysulfides were investigated at pH 9 in methanol/water solutions at two different temperatures (25 °C and 40 °C). Under all conditions investigated, α-HBCDD reacts 10 to 20 times slower with Bisulfide than β-HBCDD and γ-HBCDD. The difference in reactivity of HBCDD isomers can be explained by the different populations of stable conformers with large dihedral angle between the vicinal bromine atoms. It was also observed that the reaction of HBCDD with polysulfides is about six times faster than with Bisulfide. The experiments performed in solvent mixtures with increased water content at 40 °C indicated that the reaction of HBCDD with Bisulfide is faster with higher percentage of water. The much slower abiotic reaction of α-HBCDD compared to β-HBCDD and γ-HBCDD could potentially contribute to the fact that α-HBCDD is more persistent in the environment than γ-HBCDD. Only one isomer of tetrabromocyclododecene (TBCDe-5) was identified as a degradation product of the reaction of HBCDD with reduced sulfur species. TBCDe-5 itself reacts about ten times slower with Bisulfide and twenty times slower with polysulfide than HBCDD. The study demonstrates that polysulfides and Bisulfides can reduce HBCDD sufficiently in natural anoxic environments and the dominant pathway for the degradation of HBCDD by reduced sulfur species is very likely to be the reductive debromination of vicinal dibromides via concerted anti-elimination.
-
nucleophilic reactions of phorate and terbufos with reduced sulfur species under anoxic conditions
Journal of Agricultural and Food Chemistry, 2007Co-Authors: And Qiu Gan, Urs JansAbstract:The reactions of phorate and terbufos with Bisulfide (HS-), polysulfide (Sn2-), thiosulfate (S2O32-), and thiophenolate (PhS-) were examined in well-defined aqueous solution under anoxic conditions to investigate their role in the degradations of phorate and terbufos. Reactions were monitored at various concentrations of reduced sulfur species to obtain the second-order rate constants. The reactivity of the reduced sulfur species decreased in the order Sn2- > PhS- > HS- > S2O32-. Hydrolysis products, formaldehyde and diethyl disulfide/di-tert-butyl disulfide, indicated that OH-/H2O attacked the carbon atom between the two sulfur atoms, the so-called thioacetal carbon, which is very reactive due to the presence of the two neighboring sulfur atoms. The reaction of phorate and terbufos with PhS- was investigated to study the transformation products in the reactions with reduced sulfur species. The transformation products demonstrated that the observed increase in rate constants in the reaction with reduced s...
Harold A. Scheraga - One of the best experts on this subject based on the ideXlab platform.
-
role of the 65 72 disulfide bond in oxidative folding of bovine pancreatic ribonuclease a
Biochemistry, 2003Co-Authors: Hang Cheol Shin, Mahesh Narayan, Myeong Cheol Song, Harold A. ScheragaAbstract:To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond. There is no bias (over the entropic influence) for the three native disulfide bonds, [26-84], [40-95], and [58-110]. Previous oxidative folding results for wt-RNase A indicated the predominance of the des [40-95] intermediate over des [65-72] after the rate-determining step in the regeneration process. Considering that there is no preferential distribution of disulfides in the 1S ensemble of [C65S, C72S], in contrast to the preferential population of the [65-72] disulfide bond in wt-RNase A, these results indicate a critical role for the [65-72] disulfide bond in the regeneration of wt-RNase A. Furthermore, analysis of the disulfide distribution of the 1S intermediates of [C65S, C72S] compared to that of wt-RNase A lends support for a physicochemical basis for the previously observed slow folding rate of this mutant, compared to its analogue (des [65-72]) of wt-RNase A.
-
disulfide bonds and protein folding
Biochemistry, 2000Co-Authors: William J. Wedemeyer, Ervin Welker, Mahesh Narayan, Harold A. ScheragaAbstract:The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of “rugged funnel” models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of ...
-
kinetic folding pathway of a three disulfide mutant of bovine pancreatic ribonuclease a missing the 40 95 disulfide bond
Biochemistry, 1998Co-Authors: Harold A. ScheragaAbstract:The oxidative refolding pathway of a three-disulfide mutant of bovine pancreatic ribonuclease A (RNase A) from the fully reduced unfolded form to the native state has been studied by using oxidized and reduced dithiothreitol as the redox reagents at pH 8.0 and 25 degrees C. This mutant was prepared by replacing Cys40 and Cys95 in RNase A with alanines while maintaining the other three native disulfide bonds to mimic one of the two major three-disulfide intermediates (des-[40-95]) observed in the regeneration of wild-type RNase A. The kinetics of refolding of this mutant were measured by quenching the regeneration reaction at various times with a rapid blocking reagent, 2-aminoethyl methanethiosulfonate (AEMTS), fractionating the disulfide intermediates by using cation-exchange HPLC, and analyzing the time course of each group of disulfide species. It was found that the disulfide intermediates formed during regeneration reach a steady-state distribution after a short period of preequilibration similar to that in the regeneration of wild-type RNase A. The experimental data acquired under different redox conditions were fit to a kinetic model with a steady-state treatment. The fitted results indicate that this mutant refolds through a rate-determining step which involves the oxidation of certain two-disulfide species to form a putative three-disulfide species which proceeds rapidly to the native protein. A rough estimation suggests that this pathway could constitute no more than 5% of the major pathway leading to the formation of des-[40-95] (the major three-disulfide intermediate formed) in the regeneration of wild-type RNase A. Several kinetic constants pertaining to the oxidation and reduction of various disulfide intermediates were compared with those obtained in the regeneration studies of wild-type RNase A to gain further understanding about the folding pathways of RNase A. Comparisons are also given for the oxidative refolding studies of several other three disulfide bond proteins, suggesting that the formation of a large number of disulfide-bonded intermediates during oxidative refolding is probably a common feature for most proteins.
Masahiko Yamaguchi - One of the best experts on this subject based on the ideXlab platform.
-
equilibrium shift induced by chiral nanoparticle precipitation in rhodium catalyzed disulfide exchange reaction
Tetrahedron, 2015Co-Authors: Masamichi Miyagawa, Mieko Arisawa, Masahiko YamaguchiAbstract:Abstract Chiral silica (P)-nanoparticles grafted with (P)-helicene recognize the molecular structure of a chiral diol disulfide in the presence of monool disulfide and dibutyl disulfide. The (P)-nanoparticles selectively adsorb the diol disulfide, aggregate, and precipitate from solution. Under rhodium-catalyzed equilibrium among three disulfides, the diol disulfide is removed from solution by precipitation, which induces an equilibrium shift in the solution. By conducting the precipitation experiment twice, we obtained the diol disulfide in 37% yield from a statistical 1:2:1 equilibrium mixture of three disulfides. The method is applied to a racemic monool disulfide, and an optically active diol disulfide is obtained via kinetic resolution and equilibrium shift.
-
rhcl3 catalyzed disulfide exchange reaction using water solvent in homogeneous and heterogeneous systems
Journal of Organometallic Chemistry, 2006Co-Authors: Mieko Arisawa, Atsushi Suwa, Masahiko YamaguchiAbstract:Abstract RhCl 3 catalyzed the alkylthio exchange reaction of hydrophilic disulfides in water under homogeneous conditions, and equilibrium was attained in several hours. The reaction was applied to the exchange of unprotected glutathione disulfide. The reaction of dimethyl disulfide and hydrophilic disulfides under heterogeneous conditions also proceeded effectively. The mechanism turned out to be dependent on the water solubility of the substrates: The reaction of bis(3-hydroxypropyl) disulfide took place in the dimethyl disulfide phase, whereas the reaction of bis(6-aminohexyl) disulfide dihydrochloride proceeded in the water phase.
-
rhodium catalyzed disulfide exchange reaction
Journal of the American Chemical Society, 2003Co-Authors: Mieko Arisawa, Masahiko YamaguchiAbstract:A system of RhH(PPh3)4, trifluoromethanesulfonic acid, and (p-tol)3P catalyzes the disulfide exchange reaction. Treatment of two symmetrical dialkyl disulfides with the catalyst provides an equilibrium mixture of three disulfides within 15 min in refluxing acetone. The catalyst is active after reaching the equilibrium, and addition of a disulfide to the mixture changes the ratio of the products. The use of 4 mol equiv excess of one of the disulfides provides the unsymmetrical disulfide in a yield exceeding 80%. Disulfide-containing peptides also undergo an exchange reaction. The reactions of diaryl disulfides and dialkyl disulfides are even faster, and reach equilibrium within 5 min at room temperature in the presence of the rhodium complex and 1,2-bis(diphenylphosphino)ethane (dppe). This exchange reaction is considerably affected by the substituents on the disulfides. Treatment of diphenyl disulfide, di(p-tolyl) disulfide, and bis(sec-butyl) disulfide yields phenyl p-tolyl disulfide at room temperature ...
Neil J. Bulleid - One of the best experts on this subject based on the ideXlab platform.
-
folding of a single domain protein entering the endoplasmic reticulum precedes disulfide formation
Journal of Biological Chemistry, 2017Co-Authors: Philip Robinson, Marie Anne Pringle, Cheryl A Woolhead, Neil J. BulleidAbstract:The relationship between protein synthesis, folding, and disulfide formation within the endoplasmic reticulum (ER) is poorly understood. Previous studies have suggested that pre-existing disulfide links are absolutely required to allow protein folding and, conversely, that protein folding occurs prior to disulfide formation. To address the question of what happens first within the ER, that is, protein folding or disulfide formation, we studied folding events at the early stages of polypeptide chain translocation into the mammalian ER using stalled translation intermediates. Our results demonstrate that polypeptide folding can occur without complete domain translocation. Protein disulfide isomerase (PDI) interacts with these early intermediates, but disulfide formation does not occur unless the entire sequence of the protein domain is translocated. This is the first evidence that folding of the polypeptide chain precedes disulfide formation within a cellular context and highlights key differences between protein folding in the ER and refolding of purified proteins.
-
thiol disulfide exchange between the pdi family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme
Biochemical Journal, 2015Co-Authors: Ojore B V Oka, Hui Ying Yeoh, Neil J. BulleidAbstract:The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm.
-
glutathione is required to regulate the formation of native disulfide bonds within proteins entering the secretory pathway
Journal of Biological Chemistry, 2004Co-Authors: Seema Chakravarthi, Neil J. BulleidAbstract:The formation of native disulfide bonds is an essential event in the folding and maturation of proteins entering the secretory pathway. For native disulfides to form efficiently an oxidative pathway is required for disulfide bond formation and a reductive pathway is required to ensure isomerization of non-native disulfide bonds. The oxidative pathway involves the oxidation of substrate proteins by PDI, which in turn is oxidized by endoplasmic reticulum oxidase (Ero1). Here we demonstrate that overexpression of Ero1 results in the acceleration of disulfide bond formation and correct protein folding. In contrast, lowering the levels of glutathione within the cell resulted in acceleration of disulfide bond formation but did not lead to correct protein folding. These results demonstrate that lowering the level of glutathione in the cell compromises the reductive pathway and prevents disulfide bond isomerization from occurring efficiently, highlighting the crucial role played by glutathione in native disulfide bond formation within the mammalian endoplasmic reticulum.