Disulfide bond isomerization in prokaryotes

Proteins with multiple cysteine residues often require disulfide isomerization reactions before they attain their correct conformation. In prokaryotes this reaction is catalyzed mainly by DsbC, a protein that shares many similarities in structure and mechanism to the eukaryotic protein disulfide isomerase. This review discusses the current knowledge about disulfide isomerization in prokaryotes.     

Disulfide bond formation in prokaryotes: History,diversity and design

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Disulfide bond plasticity in epidermal growth factor

Epidermal growth factor (EGF) has a (1-3,2-4,5-6) disulfide-bonding pattern. This pattern is found in nearly all EGF-like domains, despite wide variation in sequences. Biological data from EGF and at least one EGF-like domain show that disulfide bond isomers have significant bioactivity and suggests that the EGF fold can accommodate alternate disulfide-bonding patterns. The disulfide bonds in murine EGF were altered to seven different patterns and structures were calculated incorporating all the restraints from the highest resolution restraint set available (Tejero et al., 1996). Results showed that besides the native (1-3,2-4,5-6), two other disulfide-bonding patterns: (1-2,3-4,5-6) and (1-3,2-5,4-6) satisfied the restraints as well as the native. The results for these two patterns were indistinguishable from the native on the basis of distance and dihedral violations, XPLOR energies, Procheck statistics, and RMSDs of the final set of structures. Two other disulfide bond patterns, (1-2,3-5, 4-6) and (1-4,2-3,5-6) were able to satisfy all the distance restraints but had one or more cysteine dihedral violations. For all seven isomers, the final calculated structures were highly similar to EGF with all-atom RMSD's in the 1. 5-2 A range. These results suggest that the EGF backbone fold has the unique property of accommodating several different disulfide-bonding patterns.     

Disulfide bond shuffling in bovine alpha-lactalbumin: MD simulation confirms experiment

A simple and straightforward classical molecular dynamics simulation technique is proposed to predict possible disulfide bridge shuffling. Application to bovine alpha-lactalbumin shows that shuffling can be observed on short simulation time scales and yields results in agreement with experiment.     

Temperature effect on bacterial azo bond reduction kinetics: an Arrhenius plot analysis

Studied was the effect of temperature in the range 12–46 °C on the rate of bacterial decolorization of the mono-azo dye Acid Orange 7 by Alcaligenes faecalis 6132 and Rhodococcus erythropolis 24. With both strains the raise of temperature led to a corresponding raise of decolorization rate better manifested by R. erythropolis. The analysis of the Arrhenius plot revealed a break near the middle of the temperature range. The regression analysis showed practically complete identity of the observed break point temperatures (T _BP): 20.7 °C for Alc. faecalis and 20.8 °C for R. erythropolis. The values of the activation energy of the decolorization reaction (E _a) were found to depend on both the organism and the temperature range. In the range below T _BP the estimated values of E _a were 138 ± 7 kJ mol⁻¹ for Alc. faecalis and 160 ± 8 kJ mol⁻¹ for R. erythropolis. In the range above T _BP they were 54.2 ± 1.8 kJ mol⁻¹ for Alc. faecalis and 37.6 ± 4.1 kJ mol⁻¹ for R. erythropolis. Discussed are the possible reasons for the observed abrupt change of the activation energy.     

Disulfide bond generation in mammalian blood serum: detection and purification of quiescin-sulfhydryl oxidase

A sensitive new plate-reader assay has been developed showing that adult mammalian blood serum contains circulating soluble sulfhydryl oxidase activity that can introduce disulfide bonds into reduced proteins with the reduction of oxygen to hydrogen peroxide. The activity was purified 5000-fold to >90% homogeneity from bovine serum and found by mass spectrometry to be consistent with the short isoform of quiescin-sulfhydryl oxidase 1 (QSOX1). This FAD-dependent enzyme is present at comparable activity levels in fetal and adult commercial bovine sera. Thus cell culture media that are routinely supplemented with either fetal or adult bovine sera will contain this facile catalyst of protein thiol oxidation. QSOX1 is present at approximately 25 nM in pooled normal adult human serum. Examination of the unusual kinetics of QSOX1 toward cysteine and glutathione at low micromolar concentrations suggests that circulating QSOX1 is unlikely to significantly contribute to the oxidation of these monothiols in plasma. However, the ability of QSOX1 to rapidly oxidize conformationally mobile protein thiols suggests a possible contribution to the redox status of exofacial and soluble proteins in blood plasma. Recent proteomic studies showing that plasma QSOX1 can be utilized in the diagnosis of pancreatic cancer and acute decompensated heart failure, together with the overexpression of this secreted enzyme in a number of solid tumors, suggest that the robust QSOX assay developed here may be useful in the quantitation of enzyme levels in a wide range of biological fluids.     

Disulfide bond cross-linked dimer in acetylcholine receptor from Torpedo californica

Acetylcholine receptor from $\text{Torpedo californica}$ electric tissue occurs in membrane, and is purified, as a mixture of monomer and dimer. Dimer is cross-linked by disulfide bonds involving one of the four polypeptide components of receptor, namely the one of apparent molecular weight of 64,000.     

Disulfide bond effects on protein stability: designed variants of Cucurbita maxima trypsin inhibitor-V

Attempts to increase protein stability by insertion of novel disulfide bonds have not always been successful. According to the two current models, cross-links enhance stability mainly through denatured state effects. We have investigated the effects of removal and addition of disulfide cross-links, protein flexibility in the vicinity of a cross-link, and disulfide loop size on the stability of Cucurbita maxima trypsin inhibitor-V (CMTI-V; 7 kD) by differential scanning calorimetry. CMTI-V offers the advantage of a large, flexible, and solvent-exposed loop not involved in extensive intra-molecular interactions. We have uncovered a negative correlation between retention time in hydrophobic column chromatography, a measure of protein hydrophobicity, and melting temperature (T(m)), an indicator of native state stabilization, for CMTI-V and its variants. In conjunction with the complete set of thermodynamic parameters of denaturation, this has led to the following deductions: (1) In the less stable, disulfide-removed C3S/C48S (Delta Delta G(d)(50 degrees C) = -4 kcal/mole; Delta T(m) = -22 degrees C), the native state is destabilized more than the denatured state; this also applies to the less-stable CMTI-V* (Delta Delta G(d)(50 degrees C) = -3 kcal/mole; Delta T(m) = -11 degrees C), in which the disulfide-containing loop is opened by specific hydrolysis of the Lys(44)-Asp(45) peptide bond; (2) In the less stable, disulfide-inserted E38C/W54C (Delta Delta G(d)(50 degrees C) = -1 kcal/mole; Delta T(m) = +2 degrees C), the denatured state is more stabilized than the native state; and (3) In the more stable, disulfide-engineered V42C/R52C (Delta Delta G(d)(50 degrees C) = +1 kcal/mole; Delta T(m) = +17 degrees C), the native state is more stabilized than the denatured state. These results show that a cross-link stabilizes both native and denatured states, and differential stabilization of the two states causes either loss or gain in protein stability. Removal of hydrogen bonds in the same flexible region of CMTI-V resulted in less destabilization despite larger changes in the enthalpy and entropy of denaturation. The effect of a cross-link on the denatured state of CMTI-V was estimated directly by means of a four-state thermodynamic cycle consisting of native and denatured states of CMTI-V and CMTI-V*. Overall, the results show that an enthalpy-entropy compensation accompanies disulfide bond effects and protein stabilization is profoundly modulated by altered hydrophobicity of both native and denatured states, altered flexibility near the cross-link, and residual structure in the denatured state.     

Disulfide reduction and sulfhydryl uptake by Streptococcus mutans

Incubation of Streptococcus mutans cells with certain disulfide compounds resulted in accumulation of reduced sulfhydryl compounds in the extracellular medium or in both the medium and the cells. Oxidized lipoic acid and lipoamide competed for reduction. At high concentrations, these compounds were reduced at rates comparable to that of glucose metabolism, and all of the increase in sulfhydryls was in the medium. Cystamine did not compete with these compounds for reduction but was also reduced at high rates and low apparent affinity, and all of the cysteamine produced from cystamine accumulated in the medium. In contrast, glutathione disulfide (GSSG) and L-cystine were reduced slowly but with high apparent affinity, and 60 to 80% of the increase in sulfhydryls was intracellular. NADH-dependent lipoic acid or lipoamide reductase activity was present in the particulate (wall-plus-membrane) fraction, whereas NADPH-dependent GSSG reductase activity was present in the soluble (cytoplasmic) fraction. Two transport systems for disulfide and sulfhydryl compounds were distinguished. GSSG, L-cystine, and reduced glutathione competed for uptake. L-Cysteine was taken up by a separate system that also accepted L-penicillamine and D-cysteine as substrates. Uptake of glutathione or L-cysteine, or the uptake and reduction of GSSG or L-cystine, resulted in up to a 10-fold increase in cell sulfhydryl content that raised intracellular concentrations to between 30 and 40 mM. These reductase and transport systems enable S. mutans cells to create a reducing environment in both the extracellular medium and the cytoplasm.     

Disulfide bond within mu-calpain active site inhibits activity and autolysis

Oxidative processes have the ability to influence micro-calpain activity. In the present study the influence of oxidation on activity and autolysis of micro-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of micro-calpain is diminished by oxidation with H(2)O(2) in a reversible manner involving cysteine and that the rate of autolysis of micro-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized micro-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in micro-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for micro-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.     

Disulfide bond formation,a race between FAD and oxygen

The long-running race to find the source of oxidizing potential for disulfide bond formation is over. The winner is one of the first contestants to enter: oxygen.     

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.

The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.     

Clotting of fibrinogen. 1. Scanning calorimetric study of the effect of calcium

The denaturation temperature Td and the enthalpy of thermal denaturation delta Hd of the D nodules of fibrinogen increase 12-13 degrees C and 40%, respectively, when fibrinogen is clotted by thrombin in the presence of 10(-3) M calcium ion. The rate of change of Td and delta Hd is first order in thrombin concentration. In the absence of calcium, little change in Td is observed, but the increase in delta Hd still occurs. The shift in Td as a function of logarithm of calcium concentration is sigmoid, with a half-point at 2.5 X 10(-5) M calcium for human and 6.0 X 10(-5) M calcium for bovine fibrinogens, suggesting that the shift is due to binding of calcium at the high-affinity binding sites of fibrin. The Td of the D nodule of native fibrinogen also increases, but not as much, on addition of calcium. This increase in Td is also sigmoid with log calcium, with a half-point of 1.6 X 10(-3) M calcium for human and 3.2 X 10(-3) M calcium for bovine fibrinogens, and appears to be due to binding of calcium to the low-affinity binding sites of fibrinogen. At calcium concentrations greater than 10(-4) M, traces of factor XIII in the bovine fibrinogen preparation become activated and cause cross-linking of the fibrin gel. But the changes in Td and delta Hd still occur when factor XIIIa is inactivated by iodoacetamide, and the rate of the changes is not altered by addition of large amounts of factor XIIIa.(ABSTRACT TRUNCATED AT 250 WORDS)     

The binding of calcium to fibrinogen: influence on the clotting process.

The influence of Ca2+ on the basic reaction between thrombin and fibrinogen was investigated. The results demonstrate that: (a) A Ca2+-dependent dimeric intermediate is formed during the early step of the clotting process. This dimeric intermeidate is shown to be formed by the association of an intact fibrinogen molecule and a fibrin monomer devoid in only the peptide A, (b) Ca2+ enhances the proteolytic step as illustrated by the measure of the kinetics of H+ release at pH 8.6. On the basis of these observations it is proposed that Ca2+ catalyses the proteolysis of fibrinogen by thrombin through the formation of a Ca2+-dependent dimer.     

Disulfide bond formation in the human immunodeficiency virus type 1 Nef protein.

Substitution of alanine for cysteine residues of the human immunodeficiency virus type 1 LAI (BRU) and ELI Nef proteins was used to determine pairing of the cysteine residues present in each protein. The results show that under nonreducing conditions, alternative pairing of the cysteines occurs. The preferred pairing of cysteine residues of the LAI and ELI proteins differs. In the experimental system used, viruses carrying the ELI nef allele are found to express Nef proteins which accelerate virus replication. Mutation in critical cysteine residues of the protein reduce the rate of virus replication. In the same system, viruses harboring the LAI nef allele fail to replicate. These observations raise the possibility that differences in the observed biological activity of nef alleles may be attributed, at least in part, to differences in the secondary structure of the proteins.