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Kinetic and thermodynamic properties of two barley thioredoxin h isozymes, HvTrxh1 and HvTrxh2
FEBS Letters
Abstract
Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were approximately 60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the active site Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacetamide titration, but showed approximately 70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes.
... Increases in the insulin concentration result in a classical substrate saturation profile and Michaelis-Menten parameters have consequently been assigned to thioredoxin. 13,[15][16][17][18][19] Despite mammalian insulin not being the physiological substrate for all the thioredoxins characterised with this assay, it is readily reduced by most thioredoxins. This common substrate therefore allows for semi-quantitative kinetic comparisons between different thioredoxins. ...
... The insulin reduction assay is one of the most commonly used methods to determine thioredoxin activity in vitro, but the classical substrate saturation profile generated with increasing insulin concentrations has usually been fitted to the Michaelis-Menten equation (Scheme I), 13,[15][16][17][18][19] although we recently proposed that these datasets should be fitted to a model of the entire system of reactions (i.e. the redox couple model approach, cf. Scheme II). ...
Introduction:
The thioredoxin system, consisting of thioredoxin reductase, thioredoxin and NADPH, is present in most living organisms and reduces a large array of target protein disulfides.
Objective:
The insulin reduction assay is commonly used to characterise thioredoxin activity in vitro, but it is not clear whether substrate saturation datasets from this assay should be fitted and modeled with the Michaelis-Menten equation (thioredoxin enzyme model), or fitted to the thioredoxin system with insulin reduction described by mass-action kinetics (redox couple model).
Methods:
We utilized computational modeling and in vitro assays to determine which of these approaches yield consistent and accurate kinetic parameter sets for insulin reduction.
Results:
Using computational modeling, we found that fitting to the redox couple model, rather than to the thioredoxin enzyme model, resulted in consistent parameter sets over a range of thioredoxin reductase concentrations. Furthermore, we established that substrate saturation in this assay was due to the progressive redistribution of the thioredoxin moiety into its oxidised form. We then confirmed these results in vitro using the yeast thioredoxin system.
Discussion:
This study shows how consistent parameter sets for thioredoxin activity can be obtained regardless of the thioredoxin reductase concentration used in the insulin reduction assay, and validates computational systems biology modeling studies that have described the thioredoxin system with the redox couple modeling approach.
... In cereal seeds, reduction of disulfides in proteinaceous hydrolase inhibitors by h-type Trx was proposed to facilitate starch mobilisation [15]. Structural and biochemical properties of barley (Hordeum vulgare) Trxh (HvTrxh) have been characterised [16][17][18][19][20] and a large number of potential target proteins were identified using proteomics approaches, including a-amylase inhibitors of the CM-protein family [21][22][23][24]. ...
... Treatment (1 h) of LDI with HvTrxh1 or HvTrxh2 released on average 5.3 and 4.0, respectively, of a possible maximum of 10 thiol groups (Fig. 3, insert), reflecting the reported higher reactivity of HvTrxh1 [20]. Longer incubation (9 h) caused essentially complete reduction of all LDI disulfide bonds, HvTrxh1/HvTrxh2 releasing 9.8/9.2 ...
Barley limit dextrinase (LD) that catalyses hydrolysis of α-1,6 glucosidic linkages in starch-derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM-protein family) and has four disulfide bridges and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function.
... Since Trxs seem to recognize target proteins based on threedimensional structures rather than sequence-specific regions on target proteins, the understanding of the interactions between Trx, target proteins and the Trx reductases requires structural information about the interacting partners [64,65]. These studies have been complemented by mutational, kinetic or thermodynamic studies [66][67][68][69][70][71]. Despite the wide variety of products and the diversity in the biological outcome of disulfide reduction that results from the activity of individual Trxs, all types of Trx h share a similar fold, known as the Trx-fold. ...
Thioredoxins are ubiquitous disulfide reductases involved in a wide range of biochemical pathways in various biological systems, and also implicated in numerous biotechnological applications. Plants uniquely synthesize an array of thioredoxins targeted to different cell compartments, for example chloroplastic f- and m-type thioredoxins involved in regulation of the Calvin-Benson cycle. The cytosolic h-type thioredoxins act as key regulators of seed germination and are recycled by NADPH-dependent thioredoxin reductase. The present review on thioredoxin h systems in plant seeds focuses on occurrence, reaction mechanisms, specificity, target protein identification, three-dimensional structure and various applications. The aim is to provide a general background as well as an update covering the most recent findings.
... 59 Like EcTrxR, HvNTR2 shows a low rate of O 2 reduction, observed as a background rate in measurements of insulin reduction by the barley Trxs. 60 Apart from the accessibility of O 2 , stabilization of the activated oxygen species in the reaction intermediate greatly affects the rate, e.g., by a lysine residue, identified to be primarily responsible for stabilization of the transient negative charge on FAD C(4a)-hydroperoxide in sarcosine oxidase. 2,61 At present, LMW TrxR structures in the FO conformation from 14 species are available in the Protein Data Bank (PDB). ...
Thioredoxin, involved in numerous redox pathways, is maintained in the dithiol state by the NADPH-dependent flavoprotein thioredoxin reductase (TrxR). Here, TrxR from Lactococcus lactis is compared with the well-characterized TrxR from Escherichia coli. The two enzymes belong to the same class of low molecular weight thioredoxin reductases and display similar kcat (~25 s(-1)) with their cognate thioredoxin. Remarkably, however, the L. lactis enzyme is inactivated by visible light and furthermore reduces molecular oxygen 10 times faster than E. coli TrxR. The rate of light inactivation at standardized conditions (λmax=460 nm and 4 °C) was reduced at lowered oxygen concentration and in the presence of iodide. Inactivation was accompanied by a distinct spectral shift of the FAD that remained firmly bound. High resolution mass spectrometric analysis of heat extracted FAD from light-damaged TrxR revealed a mass increment of 13.979 Da, relative to unmodified FAD, corresponding to the addition of one oxygen atom and loss of two hydrogen atoms. Tandem mass spectrometry confined the mass increase to the isoalloxazine ring and the extracted modified cofactor reacted with dinitrophenyl hydrazine indicating the presence of an aldehyde. We hypothesize that a methyl group of FAD is oxidized to a formyl group. The significance of this not previously reported oxidation and the exceptionally high rate of oxygen-reduction are discussed in relation to other flavin-modifications and possible occurrence of enzymes with similar properties.
... Several studies attempted to evaluate the affinities of Trx to its target proteins or the reduction efficiencies of the target proteins by determining K m or k cat /K m values obtained from the results of complete reduction of the target proteins using Trx (Collin et al., 2003;Perez-Perez et al., 2009;Maeda et al., 2010). However, a rather enigmatic behavior of Trx remained unresolved; the reduced form of Trx preferentially interacts with the oxidized form of the target protein and the oxidized form of Trx must release the reduced form of the target in order to ensure an efficient reduction cycle. ...
Thioredoxin is a critical protein that mediates the transfer of reducing equivalents in vivo and regulates redox sensitive enzymes in several cases. In addition, thioredoxin provides reducing equivalents to oxidoreductases such as peroxiredoxin. Through a dithiol–disulfide exchange reaction, the reduced form of thioredoxin preferentially interacts with the oxidized forms of targets, which are immediately released after this reaction is complete. In order to more thoroughly characterize these interactions between thioredoxin and its target proteins, a mutant version of thioredoxin that lacked the second cysteine was synthesized and interactions were monitored by surface plasmon resonance. The binding rates of thioredoxin to its targets were very different depending on the use of reducing equivalents by the targets: the enzymes whose activity was controlled by reduction or oxidation of a cysteine pair(s) in the molecule and the enzymes that used reducing equivalents provided by thioredoxin for their catalysis. In addition, thioredoxin revealed a stronger preference for an oxidized target. These results explain the reason for selective association of thioredoxin with oxidized targets for reduction, whereas immediate dissociation from a reduced target when the dithiol–disulfide exchange reaction is complete.
... HvTrxh1 and HvTrxh2 show 51% sequence identity and similar biophysical characteristics. The redox potentials (E @BULLET@BULLET ) of both proteins was determined to be −270 mV in a fluorometric assay using Escherichia coli Trx as a reference and the pK a of the nucleophilic active site thiol (CGPC) in both HvTrxh1 and HvTrxh2 were determined to be 7.6 by iodoacetamide (IAM) alkylation kinetics (Maeda et al., 2010). Nevertheless, HvTrxh1 displays slightly higher thiol reactivity and higher affinity for the model substrate insulin, possibly due to subtle differences in the local environment surrounding the active site. ...
Thioredoxin (Trx) reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type Trx facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent Trx reductase. This review presents a summary of the research conducted during the last 10 years to elucidate the structure and function of the barley seed Trx system at the molecular level combined with proteomic approaches to identify target proteins.
... Plants have multiple isoforms of trx h, and there are two barley trx h isoforms and three wheat trx h isoforms to be reported [25,26]. Trx h had an important role in the germination of cereals (wheat and barley), however, the two barley trx h isoforms and the three wheat trx h isoforms showed distinct expression patterns in germinating seeds, indicating they had different role in the germination of cereal seeds [27]. ...
Thioredoxin h can regulate the redox environment in the cell and play an important role in the germination of cereals. In the present study, the thioredoxin s antisense transgenic wheat with down-regulation of thioredoxin h was used to study the role of thioredoxin h in protein metabolism during germination of wheat seeds, and to explore the mechanism of the thioredoxin s antisense transgenic wheat seeds having high resistance to pre-harvest sprouting. The qRT-PCR results showed that the expression of protein disulfide isomerase in the thioredoxin s antisense transgenic wheat was up-regulated, which induced easily forming glutenin macropolymers and the resistance of storage proteins to degradation. The expression of serine protease inhibitor was also up-regulated in transgenic wheat, which might be responsible for the decreased activity of thiocalsin during the germination. The expression of WRKY6 in transgenic wheat was down-regulated, which was consistent with the decreased activity of glutamine oxoglutarate aminotransferase. In transgenic wheat, the activities of glutamate dehydrogenase, glutamic pyruvic transaminase and glutamic oxaloacetic transaminase were down-regulated, indicating that the metabolism of amino acid was lower than that in wild-type wheat during seed germination. A putative model for the role of thioredoxin h in protein metabolism during wheat seed germination was proposed and discussed.
The acidity of small alkylthiols depends mainly on the inductive effect of substituents, and the ionization state of neighbor functional groups. Protein thiols have a wide range of pKas, that depend, qualitatively on the electrostatic environment, due to charged neighboring groups and α-helix macrodipoles, hydrogen bonds. The desolvation of the thiol/thiolate group also causes major alterations of the pKa.
Nucleophilicity is the most important kinetic property of the thiolate catalytic cysteines in proteins, it is not related to pKa and depends on the regulation of the solvation as well as hydrogen bonding of protein thiolates. In general, aqueous environments greatly diminish the nucleophilicity of thiolates but the protein environment can delicately modulate it by tweaking its nonbonding and solvation interactions, and importantly enhancing its reactivity when the right substrate is bound and ready to react, this explains why protein thiolate reactivity is exquisitely selective.
Free thiol-containing proteins are suggested to work as antioxidants in beer, but the majority of thiols in wort are present in their oxidized form as disulfides and are therefore not active as antioxidants. Thioredoxin, a disulfide-reducing protein, is released into the wort from some yeast strains during fermentation. The capacity of the thioredoxin enzyme system (thioredoxin, thioredoxin reductase, NADPH) to reduce oxidized thiols in boiled wort under fermentation-like conditions were studied. Free thiols were quantitated in boiled wort samples by derivatization with ThioGlo®1 and fluorescence detection of thiol-derivatives. When boiled wort was incubated with all components of the thioredoxin system at pH 7.0 and 25 C for 60 min under anaerobic conditions, the free thiol concentration increased from 25 to 224 µM. At pH values similar to wort (pH 5.7) and beer (pH 4.5), the thioredoxin system was also capable of increasing the free thiol concentration, although with lower efficiency to 187 and 170 µM, respectively. The presence of sulfite, an important antioxidant in beer secreted by the yeast during fermentation, was found to inactivate thioredoxin by sulfitolysis. Reduction of oxidized thiols by the thioredoxin system was therefore only found to be efficient in the absence of sulfite.
Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, the Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of ten nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In this study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to ten Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe-S cluster. By reconstituting the FTR/Trx system in vitro , we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4'-maleimidylstilbene-2,2'-disulfonate (AMS), indicated that all of chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.
The cellular redox states directly affect cell proliferation, differentiation and apoptosis, and the redox states changes is particularly important to the regulation of cell survival or death. Thioredoxin is a kind of oxidation regulatory protein which is widely exists in organisms, and the change of redox states is also an important process in redox regulation. In this work, we have used the site-directed mutagenesis of protein, SDS-polyacrylamide gel electrophoresis fluorescence spectroscopy and circular dichroism etc., to investigate redox states changes between TRX (E. coli) and glutathione peroxidase(GPX3) during their interaction. By observing the fluorescence spectra of TRX and its mutants, we have studied the protein interactions as well as the redox states switching between oxidation state TRX and the reduced state GPX3. The results demonstrate the presence of interactions and electron exchanges occurring between reduced GPX3 and oxidized TRX, which is of significance for revealing the physical and chemical mechanism of TRX in intracellular signal transduction.
Thiol-disulfide oxidoreductases from the thioredoxin (Trx) family of proteins have a broad range of well documented functions and possess distinct substrate specificities. The mechanisms and characteristics that control these specificities are key to the understanding of both the reduction of catalytic disulfides as well as allosteric disulfides (thiol switches). Here, we have used the catalytic disulfide of E. coli 3′-phosphoadenosine 5′-phosphosulfate (PAPS) reductase (PR) that forms between the single active site thiols of two monomers during the reaction cycle as a model system to investigate the mechanisms of Trx and Grx protein specificity. Enzyme kinetics, ΔE′0 determination, and structural analysis of various Trx and Grx family members suggested that the redox potential does not determine specificity nor efficiency of the redoxins as reductant for PR. Instead, the efficiency of PR with various redoxins correlated strongly to the extent of a negative electric field of the redoxins reaching into the solvent outside the active site, and electrostatic and geometric complementary contact surfaces. These data suggest that, in contrast to common assumption, the composition of the active site motif is less important for substrate specificity than other amino acids in or even outside the immediate contact area.
Three protein disulfide reductases of the thioredoxin superfamily from the industrially important Gram-positive Lactococcus lactis (LlTrxA, LlTrxD and LlNrdH) are compared to the “classical” thioredoxin from Escherichia coli (EcTrx1). LlTrxA resembles EcTrx1 with a WCGPC active site motif and other key residues conserved. By contrast, LlTrxD is more distantly related and contains a WCGDC motif. Bioinformatics analysis suggests that LlTrxD represents a subgroup of thioredoxins from Gram-positive bacteria. LlNrdH is a glutaredoxin-like electron donor for ribonucleotide reductase class Ib. Based on protein–protein equilibria LlTrxA (E°′ = −259 mV) and LlNrdH (E°′ = −238 mV) show approximately 10 mV higher standard state redox potentials than the corresponding E. coli homologues, while E°′ of LlTrxD is −243 mV, more similar to glutaredoxin than “classical” thioredoxin. EcTrx1 and LlTrxA have high capacity to reduce insulin disulfides and their exposed active site thiol is alkylated at a similar rate at pH 7.0. LlTrxD on the other hand, is alkylated by iodoacetamide at almost 100 fold higher rate and shows no activity towards insulin disulfides. LlTrxA, LlTrxD and LlNrdH are all efficiently reduced by NADPH dependent thioredoxin reductase (TrxR) from L. lactis and good cross-reactivity towards E. coli TrxR was observed with LlTrxD as the notable exception.
Thioredoxin reduces disulfide bonds thus regulating activities of target proteins in various biological systems, e.g. inactivation of inhibitors of starch hydrolases and proteases in germinating plant seeds. In the three-dimensional structure of a complex with barley α-amylase/subtilisin inhibitor (BASI) two loops in barley thioredoxin h2 (HvTrxh2), containing an invariant cis-proline ((86)EAMP(89)) and a conserved glycine ((104)VGA(106)), surround the active site cysteines ((45)WCGPC(49)), and contribute to binding of BASI through backbone-backbone hydrogen bonds [Maeda, K., Hägglund, P., Finnie, C., Svensson, B., and Henriksen, A. (2006) Structure 14, 1701-1710]. The present study involves mutational analysis of key amino acid residues from these two loops in reactions with three protein disulfide substrates, BASI, barley glutathione peroxidase and bovine insulin as well as with NADPH-dependent barley thioredoxin reductase. HvTrxh2 M88G and M88A adjacent to the invariant cis-proline lost efficiency in both BASI disulfide reduction and recycling by thioredoxin reductase. These effects were further pronounced in M88P lacking a backbone NH group. Remarkably HvTrxh2 E86R in the same loop displayed overall retained catalytic properties, with the exception of three-fold increased activity towards BASI. From the (104)VGA(106) loop a backbone hydrogen bond donated by A106 appears important for target disulfide recognition as A106P lost 90% activity towards BASI, but was efficiently recycled by thioredoxin reductase. The findings support important roles in target recognition of backbone-backbone hydrogen bond and electrostatic interactions and are discussed in relation to earlier structural and functional studies of thioredoxins and related proteins.
Peroxiredoxins (Prx) are central elements of the antioxidant defense system and the dithiol-disulfide redox regulatory network of the plant and cyanobacterial cell. They employ a thiol-based catalytic mechanism to reduce H2O2, alkylhydroperoxide, and peroxinitrite. In plants and cyanobacteria, there exist 2-CysPrx, 1-CysPrx, PrxQ, and type II Prx. Higher plants typically contain at least one plastid 2-CysPrx, one nucleo-cytoplasmic 1-CysPrx, one chloroplast PrxQ, and one each of cytosolic, mitochondrial, and plastidic type II Prx. Cyanobacteria express variable sets of three or more Prxs. The catalytic cycle consists of three steps: (i) peroxidative reduction, (ii) resolving step, and (iii) regeneration using diverse electron donors such as thioredoxins, glutaredoxins, cyclophilins, glutathione, and ascorbic acid. Prx proteins undergo major conformational changes in dependence of their redox state. Thus, they not only modulate cellular reactive oxygen species- and reactive nitrogen species-dependent signaling, but depending on the Prx type they sense the redox state, transmit redox information to binding partners, and function as chaperone. They serve in context of photosynthesis and respiration, but also in metabolism and development of all tissues, for example, in nodules as well as during seed and fruit development. The article surveys the current literature and attempts a mostly comprehensive coverage of present day knowledge and concepts on Prx mechanism, regulation, and function and thus on the whole Prx systems in plants.
Whereas vertebrates possess only two thioredoxin genes, higher plants present a much greater diversity of thioredoxins. For example, Arabidopsis thaliana has five cytoplasmic thioredoxins (type h) and at least as many chloroplastic thioredoxins. The abundance of plant thioredoxins leads to the question whether the various plant thioredoxins play a similar role or have specific functions. Because most of these proteins display very similar activities on artificial or biological substrates in vitro, we developed an in vivo approach to answer this question. The disruption of both of the two Saccharomyces cerevisiae thioredoxin genes leads to pleiotropic effects including methionine auxotrophy, H2O2 hypersensitivity, altered cell cycle characteristics, and a limited ability to use methionine sulfoxide as source of methionine. We expressed eight plant thioredoxins (six cytoplasmic and two chloroplastic) in yeast trx1, trx2 double mutant cells and analyzed the different phenotypes. Arabidopsis type h thioredoxin 2 efficiently restored sulfate assimilation whereas Arabidopsis type h thioredoxin 3 conferred H2O2 tolerance. All thioredoxins tested could complement for reduction of methionine sulfoxide, whereas only type h thioredoxins were able to complement the cell cycle defect. These findings clearly indicate that specific interactions between plant thioredoxins and their targets occur in vivo.
The reduction, by thiol-disulfide exchange, of small model disulfides and insulin catalyzed by thioredoxin was determined spectrophotometrically from the oxidation of NADPH in the presence of thioredoxin reductase. The rate of reduction is variable due to the micro-environment of the disulfides. Cystine is reduced faster than the basic cystamine. The two interchain disulfides of bovine insulin and the corresponding S-S bonds of proinsulin were exceptionally good substrates for thioredoxin when compared with L-cystine. The K(m) value for insulin was 11 μM, and the pH optimum was 7.5. With 8 nM thioredoxin, the disulfides of insulin were reduced at a rate of 1.5 μM per min, equivalent to a turnover number for thioredoxin of 740 per min. The second order rate constant at pH 7.0 for oxidation of thioredoxin-(SH)2 with insulin disulfides was estimated from fluorescence measurements and the enzymatic rates to be around 1 x 105 M-1S-1. This is around 10,000 times higher than the second order rate constant for the reaction between insulin and dithiothreitol, demonstrating the high affinity of insulin for thioredoxin-(SH)2. Nondisulfide oxidants such as Cu2+ ions, diamide, and vitamin K3, which are known to mimic the effects of insulin on cellular hexose transport, were rapidly reduced by the thioredoxin system. The exceptional reactivity of insulin and thioredoxin-(SH)2, in vitro, is consistent with a physiological function for mammalian thioredoxin in the degradation of insulin. Functions for thioredoxin in the 'thiol oxidation-reduction model' of insulin action are discussed.
To mimic the active sites (Trp-Cys-Gly-His-Cys) contained in two thioredoxin-like domains of the eukaryotic enzyme protein disulfide-isomerase (PDI, EC 5.3.4.1), the Pro-34 residue of Escherichia coli thioredoxin (Trx) was replaced by His using site-directed mutagenesis. The mutant P34H Trx was isolated in high yield and was stable. The equilibrium between Trx and NADPH in the thioredoxin reductase (TR)-catalyzed reaction revealed that the redox potential (E'o) or P34H Trx at pH 7.0 was -235 mV as compared with -270 mV for wild type (wt) Trx. The higher E'o value made P34H Trx more similar to PDI and contributed to prominent changes in Trx functions, e.g. improved activity with TR and slower reduction of protein disulfides. Compared to wt Trx, the P34H oxidized Trx was about twice as good a substrate for TR from E. coli and four times as efficient with calf thymus TR. A novel fluorimetric assay permitted direct recording of the reaction between insulin disulfide(s) and reduced Trx. At pH 8 and 15 degrees C, second-order rate constants for wt Trx of 2 x 10(4) M-1 s-1 and for P34H Trx of 3 x 10(3) M-1 s-1 were obtained, and a different equilibrium was observed consistent with differences in E'o values. Also when the reduction mechanism of insulin was examined using NADPH and TR, P34H Trx behaved differently from wt Trx or PDI. P34H Trx may be useful as an analogue of PDI for disulfide formation in vivo and in vitro.
Fluorescence emission spectra of the oxidized and reduced forms of thioredoxin (thioredoxin-S2 and thioredoxin-(SH)2, respectively) from Escherichia coli were determined at varying pH, solvent composition, and concentration of urea or guanidine hydrochloride.
Thioredoxin-S2, with only 2 tryptophan residues at positions 28 and 31, showed a strongly quenched tryptophan fluorescence (Q = 0.02) which remained constant from pH 2 to pH 10. Addition of ethanol or dioxan up to 50% (v/v) at pH 7.0 did not perturb the tryptophan emission. Guanidine hydrochloride from 2 to 4 m increased the fluorescence intensity of thioredoxin-S2 3-fold and the emission maximum was shifted from 342 nm to 355 nm indicating unfolding of the protein. The beginning of this reversible transition was also observed in 6 m urea. Between 4 to 7 m guanidine only a small increase in tryptophan fluorescence occurred, indicating that thioredoxin-S2 was largely unfolded.
Thioredoxin-(SH)2, obtained by chemical reduction of thioredoxin-S2 by dithiothreitol, showed a pronounced pH dependence of fluorescence with a maximum at pH 5, where the emission intensity was 6.5-fold higher than that of thioredoxin-S2. The fluorescence intensity decreased on the alkaline side of pH 5 and seemed to fit a titration curve with an apparent pK of 6.75. Between 0 to 2 m guanidine hydrochloride the fluorescence intensity for thioredoxin-(SH)2 increased almost 2-fold in a transition not observed with thioredoxin-S2. Between 2 to 4 m guanidine the fluorescence intensity decreased in a transition similar to the increase in intensity for thioredoxin-S2. Under all experimental conditions, the tryptophan fluorescence intensity of thioredoxin-(SH)2 was higher than that of thioredoxin-S2, suggesting that the disulfide bond of thioredoxin-S2 was an important quenching group.
The fluorescence emission spectrum of yeast thioredoxin, which has a single tryptophan residue in a position homologous to Trp-31 of E. coli thioredoxin, showed a low quantum yield, (Q = 0.03), of tryptophan fluorescence in its oxidized form. Reduction of the disulfide bond increased the fluorescence intensity 1.2-fold. This suggests that the conformational change which accompanies reduction of thioredoxin-S2 from E. coli has its main effect on the emission of Trp-28.
Only one of the sulfhydryl groups from Cys-32 and Cys-35 in the active center of native Escherichia coli thioredoxin-(SH)2 was alkylated by excess iodoacetic acid at pH values below 8.0. Both groups reacted in the protein denatured with 4.5 M guanidine hydrochloride. The second order rate of alkylation of thioredoxin-(SH)2 with 1 eq of iodoacetic acid was pH-dependent and showed independent initial reactions of one thiolate ion with a pK value of 6.7 and a second with a pK value close to 9.0. The same pH dependence was observed for alkylation with iodoacetamide but the apparent rate constant, 107 M-1 S-1 at pH 7.2, was about 20-fold higher than the corresponding rate with iodoacetate. The sulfhydryl group with a pK value of 6.7 was shown to belong to Cys-32 by labeling thioredoxin with [14C]iodoacetic acid followed by complete alkylation with [3H]iodoacetate and amino acid sequence analysis of peptides from the active center. The abnormally low pK value of Cys-32 is suggested to arise by electrostatic influence from a positive charge on the amino group of Lys-36. A mechanism of action for thioredoxin-(SH)2 as a protein disulfide reductase has been formulated. This is based on an initial nucleophilic attack by the thiolate of Cys-32 with the formation of an unstable transient mixed disulfide involving Cys-32 and one of the sulfurs in the substrate. This is followed by a conformational change and a nucleophilic attack of Cys-35 to give the 14-membered disulfide ring in thioredoxin-S2 and the dithiol of the substrate.
Glutaredoxins belong to the thioredoxin superfamily of structurally similar thiol-disulfide oxidoreductases catalyzing thiol-disulfide exchange reactions via reversible oxidation of two active-site cysteine residues separated by two amino acids (CX1X2C). Standard state redox potential (E degrees ') values for glutaredoxins are presently unknown, and use of glutathione/glutathione disulfide (GSH/GSSG) redox buffers for determining E degrees ' resulted in variable levels of GSH-mixed disulfides. To overcome this complication, we have used reverse-phase high performance liquid chromatography to separate and quantify the oxidized and reduced forms present in the thiol-disulfide exchange reaction at equilibrium after mixing one oxidized and one reduced protein. This allowed for direct and quantitative pair-wise comparisons of the reducing capacities of the proteins and mutant forms. Equilibrium constants from pair-wise reaction with thioredoxin or its P34H mutant, which have accurately determined E degrees ' values from their redox equilibrium with NADPH catalyzed by thioredoxin reductase, allowed for transformation into standard state values. Using this new procedure, the standard state redox potentials for the Escherichia coli glutaredoxins 1 and 3, which contain identical active site sequences CPYC, were found to be E degrees ' = -233 and -198 mV, respectively. These values were confirmed independently by using the thermodynamic linkage between the stability of the disulfide bond and the stability of the protein to denaturation. Comparison of calculated E degrees ' values from a number of proteins ranging from -270 mV for E. coli Trx to -124 mV for DsbA obtained using this method with those determined using glutathione redox buffers provides independent confirmation of the standard state redox potential of glutathione as -240 mV. Determining redox potentials through direct protein-protein equilibria is of general interest as it overcomes errors in determining redox potentials calculated from large equilibrium constants with the strongly reducing NADPH or by accumulating mixed disulfides with GSH.
Thioredoxin, thioredoxin reductase and NADPH, the thioredoxin system, is ubiquitous from Archea to man. Thioredoxins, with a dithiol/disulfide active site (CGPC) are the major cellular protein disulfide reductases; they therefore also serve as electron donors for enzymes such as ribonucleotide reductases, thioredoxin peroxidases (peroxiredoxins) and methionine sulfoxide reductases. Glutaredoxins catalyze glutathione-disulfide oxidoreductions overlapping the functions of thioredoxins and using electrons from NADPH via glutathione reductase. Thioredoxin isoforms are present in most organisms and mitochondria have a separate thioredoxin system. Plants have chloroplast thioredoxins, which via ferredoxin-thioredoxin reductase regulates photosynthetic enzymes by light. Thioredoxins are critical for redox regulation of protein function and signaling via thiol redox control. A growing number of transcription factors including NF-kappaB or the Ref-1-dependent AP1 require thioredoxin reduction for DNA binding. The cytosolic mammalian thioredoxin, lack of which is embryonically lethal, has numerous functions in defense against oxidative stress, control of growth and apoptosis, but is also secreted and has co-cytokine and chemokine activities. Thioredoxin reductase is a specific dimeric 70-kDa flavoprotein in bacteria, fungi and plants with a redox active site disulfide/dithiol. In contrast, thioredoxin reductases of higher eukaryotes are larger (112-130 kDa), selenium-dependent dimeric flavoproteins with a broad substrate specificity that also reduce nondisulfide substrates such as hydroperoxides, vitamin C or selenite. All mammalian thioredoxin reductase isozymes are homologous to glutathione reductase and contain a conserved C-terminal elongation with a cysteine-selenocysteine sequence forming a redox-active selenenylsulfide/selenolthiol active site and are inhibited by goldthioglucose (aurothioglucose) and other clinically used drugs.
Oxidation-reduction midpoint potential (E(m)) versus pH profiles were measured for wild-type thioredoxins from Escherichia coli and from the green alga Chlamydomonas reinhardtii and for a number of site-directed mutants of these two thioredoxins. These profiles all exhibit slopes of approximately -59 mV per pH unit, characteristic of the uptake of two protons per reduction of an active-site thioredoxin disulfide, at acidic, neutral, and moderately alkaline pH values. At higher pH values, these profiles exhibit slopes of either -29.5 mV per pH unit, characteristic of the uptake of one proton per disulfide reduced, or are pH-independent, indicating that neither proton uptake nor proton release is associated with reduction of the active-site disulfide. Reduction of the two wild-type thioredoxins is accompanied by the uptake of two protons even at pH values where the more acidic cysteine thiol group of the reduced proteins would be expected to be completely unprotonated. The effect of site-directed mutagenesis of two highly conserved aspartate residues that play important structural and/or catalytic roles in both thioredoxins, and which could in principle play a role in proton transfer, on the pK(a) values of redox-linked acid dissociations (deduced from changes in slope of the E(m) versus pH profiles) has also been determined for both E. coli thioredoxin and C. reinhardtii thioredoxin h.
The NADPH-dependent thioredoxin reductase (NTR)/thioredoxin (Trx) system catalyzes disulfide bond reduction in the cytoplasm and mitochondrion. Trx h is suggested to play an important role in seed development, germination, and seedling growth. Plants have multiple isoforms of Trx h and NTR; however, little is known about the roles of the individual isoforms. Trx h isoforms from barley (Hordeum vulgare) seeds (HvTrxh1 and HvTrxh2) were characterized previously. In this study, two NTR isoforms (HvNTR1 and HvNTR2) were identified, enabling comparison of gene expression, protein appearance, and interaction between individual NTR and Trx h isoforms in barley embryo and aleurone layers. Although mRNA encoding both Trx h isoforms is present in embryo and aleurone layers, the corresponding proteins differed in spatiotemporal appearance. HvNTR2, but not HvNTR1, gene expression seems to be regulated by gibberellic acid. Recombinant HvNTR1 and HvNTR2 exhibited virtually the same affinity toward HvTrxh1 and HvTrxh2, whereas HvNTR2 has slightly higher catalytic activity than HvNTR1 with both Trx h isoforms, and HvNTR1 has slightly higher catalytic activity toward HvTrxh1 than HvTrxh2. Notably, both NTRs reduced Trx h at the acidic conditions residing in the starchy endosperm during germination. Interspecies reactions between the barley proteins and Escherichia coli Trx or Arabidopsis thaliana NTR, respectively, occurred with 20- to 90-fold weaker affinity. This first investigation of regulation and interactions between members of the NTR/Trx system in barley seed tissues suggests that different isoforms are differentially regulated but may have overlapping roles, with HvNTR2 and HvTrxh1 being the predominant isoforms in the aleurone layer.
The electrostatic behavior of potentially titrating groups in reduced human thioredoxin was investigated using two-dimensional (2D) 1H and 15N nuclear magnetic resonance (NMR) spectroscopy. A total of 241 chemical shift titration curves were measured over the pH range of 2.1-10.6 from homonuclear 1H-1H Hartmann-Hahn (HOHAHA) and heteronuclear 1H-15N Overbodenhausen correlation spectra. Nonlinear least-squares fits of the data to simple relationships derived from the Henderson-Hasselbalch equation led to the determination of pKas for certain isolated ionizable groups, including the single histidine residue at position 43 (pKa = 5.5 +/- 0.1) and a number of aspartic and glutamic acid carboxylate groups. Many of the titration curves demonstrate complex behavior due to the effects of interacting titrating groups, the long range of electrostatic interactions through the protein interior, and, perhaps, pH-induced conformational changes on the chemical shifts. Unambiguous assignment of the pKas for most of the 38 potentially ionizing groups of human thioredoxin could therefore not be made. In addition, there was no clear evidence that Asp-26 titrates in a manner corresponding to that observed in the Escherichia coli protein [Dyson, H. J., Tennant, L. L., & Holmgren, A. (1991) Biochemistry 30, 4262-4268]. The pKas of the active site cysteines were measured, however, with Cys-32 having an anomalously low value of 6.3 +/- 0.1 and that of Cys-35 between 7.5 and 8.6. These pKas are in agreement with proposed mechanisms for redox catalysis of thioredoxin and previously measured pKas within the active site of E. coli thioredoxin [Kallis, G. B., & Holmgren, A. (1980) J. Biol. Chem. 255, 10261-10265]. The stabilization of a thiolate anion at physiological pH can be explained by the interaction of the S gamma of Cys-32 with the amide of Cys-35 observed in the previously determined high-resolution solution structure of reduced human thioredoxin [Forman-Kay, J. D., Clore, G. M., Wingfield, P. T., & Gronenborn, A. M. (1991) Biochemistry 30, 2685-2698].
Thioltransferase, catalyzing thiol-disulfide interchange between reduced glutathione and disulfides, was purified to homogeneity from Saccharomyces cerevisiae. The purification procedure included ammonium sulfate precipitation, Sephadex G-50 gel filtration, CM-Sepharose ion exchange chromatography, and C18 reverse phase high pressure liquid chromatography. Two thioltransferase activity peaks were resolved by CM-Sepharose chromatography. The protein from the major peak had a molecular weight of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis while the minor peak protein migrated slightly faster in this gel system. Both proteins showed similar amino acid compositions and identical N-termini. The major peak of thioltransferase was extensively characterized. Plots of thioltransferase activity as a function of S-sulfocysteine or hydroxyethyl disulfide concentration did not show normal Michaelis-Menten kinetics. The enzyme activity had a pH optimum of 9.1. The protein has 106 amino acid residues with two cysteines and no arginine. The active site amino acid sequence of the enzyme was identified as Cys26-Pro-Tyr-Cys29, which is similar to that of mammalian thioltransferase and Escherichia coli glutaredoxin. The two cysteines at the active site displayed different reactivities to iodoacetamide. Cys26 was alkylated by iodoacetamide at pH 3.5 while Cys29 was alkylated at pH 8.0. The enzyme was completely inactivated when the Cys26 was carboxymethylated. A plot of incorporation of iodoacetamide into Cys29 at different pHs was similar to the pH dependence of the enzyme activity. The result suggested that Cys26 could readily initiate nucleophilic attack on disulfide substrates at physiological pH.
The thioredoxin fold is a characteristic protein structural motif that has been found in five distinct classes of proteins that have the common property of interacting with cysteine-containing substrates.
DsbA, a member of the thioredoxin family of disulfide oxidoreductases, acts in catalyzing disulfide bond formation by donating its disulfide to newly translocated proteins. We have found that the two central residues within the active site Cys-30-Pro-31-His-32-Cys-33 motif are critical in determining the exceptional oxidizing power of DsbA. Mutations that change these two residues can alter the equilibrium oxidation potential of DsbA by more than 1000-fold. A quantitative explanation for the very high redox potential of DsbA was found by measuring the pKa of a single residue, Cys-30. The pKa of Cys-30 varied dramatically from mutant to mutant and could accurately predict the oxidizing power of each DsbA mutant protein.
Within various proteins of the thioredoxin family, the stability of the disulfide bond formed reversibly between the two active site cysteine residues, one accessible and one buried, varies widely and is directly correlated with the pKa value of the accessible cysteine thiol group. If applicable to thioredoxin, its stable disulfide bond would imply that its accessible thiol group should have a high pKa value, whereas it has long been considered to be about 6.7, largely on the basis of the pH dependence of its reactivity. Such kinetic data are shown to be inconsistent with known pKa values in this case; the rate constants may reflect effects in the transition state for the reaction, which is catalyzed by thioredoxin, rather than the protein itself. Ionization of the thioredoxin thiol groups was measured indirectly by the pH dependence of the equilibrium constant for their reaction with glutathione and directly by detection of the thiolate anion by its UV absorbance. Both observations indicated that both cysteine thiol groups of thioredoxin ionize with apparent pKa values in the region of 9-10 and that their ionization is not linked strongly to that of any other groups. This conclusion is not incompatible with the other data available and would make thioredoxin consistent with the relationship between thiol group ionization and disulfide stability observed in other members of the thioredoxin family.
The active-site CXXC motif of thiol:disulfide oxidoreductases is essential for their catalysis of redox reactions. Changing the XX residues can perturb the reduction potential of the active-site disulfide bond of the Escherichia coli enzymes thioredoxin (Trx; CGPC) and DsbA (CPHC). The reduction potential is correlated with the acidity of the N-terminal cysteine residue of the CXXC motif. As the pKa is lowered, the disulfide bond becomes more easy to reduce. A change in pKa can account fully for a change in reduction potential in well-characterized CXXC motifs of DsbA but not of Trx. Formal analysis of the Nernst equation reveals that reduction potential contains both pH-dependent and pH-independent components. Indeed, the difference between the reduction potentials of wild-type Trx and wild-type DsbA cannot be explained solely by differences in thiol pKa values. Structural data for thiol:disulfide oxidoreductases reveal no single factor that determines the pH-independent component of the reduction potential. In addition, the pH-dependent component is complex when the redox state of the CXXC motif affects the titration of residues other than the thiols. These intricacies enable CXXC motifs to vary widely in their capacity to assist electron flow, and thereby engender a family of thiol:disulfide oxidoreductases that play diverse roles in biochemistry.
Thiol:disulfide oxidoreductases have a CXXC motif within their active sites. To initiate the reduction of a substrate disulfide bond, the thiolate form of the N-terminal cysteine residue (CXXC) of this motif performs a nucleophilic attack. Escherichia coli thioredoxin [Trx (CGPC)] is the best characterized thiol:disulfide oxidoreductase. Previous determinations of the active-site pKa values of Trx have led to conflicting interpretations. Here, 13C-NMR spectroscopy, site-specific isotopic labeling, and site-directed mutagenesis were used to demonstrate that analysis of the titration behavior of wild-type Trx requires the invocation of microscopic pKa values for two interacting active-site residues: Asp26 (7.5 and 9.2) and Cys32 (CXXC; 7.5 and 9.2). By contrast, in two Trx variants, D26N Trx and D26L Trx, Cys32 exhibits a pKa near 7.5 and has a well-defined, single-pKa titration curve. Similarly, in oxidized wild-type Trx, Asp26 has a pKa near 7.5. In CVWC and CWGC Trx, Cys32 exhibits a single pKa near 6.2. In all five enzymes studied here, there is no evidence for a Cys35 (CXXC) pKa of < 11. This study demonstrates that a comprehensive approach must be used to unravel complex titration behavior of the functional groups in a protein.
Two thioredoxin h isoforms, HvTrxh1 and HvTrxh2, were identified in two and one spots, respectively, in a proteome analysis of barley (Hordeum vulgare) seeds based on 2D gel electrophoresis and MS. HvTrxh1 was observed in 2D gel patterns of endosperm, aleurone layer and embryo of mature barley seeds, and HvTrxh2 was present mainly in the embryo. During germination, HvTrxh2 decreased in abundance and HvTrxh1 decreased in the aleurone layer and endosperm but remained at high levels in the embryo. On the basis of MS identification of the two isoforms, expressed sequence tag sequences were identified, and cDNAs encoding HvTrxh1 and HvTrxh2 were cloned by RT-PCR. The sequences were 51% identical, but showed higer similarity to thioredoxin h isoforms from other cereals, e.g. rice Trxh (74% identical with HvTrxh1) and wheat TrxTa (90% identical with HvTrxh2). Recombinant HvTrxh1, HvTrxh2 and TrxTa were produced in Escherichia coli and purified using a three-step procedure. The activity of the purified recombinant thioredoxin h isoforms was demonstrated using insulin and barley alpha-amylase/subtilisin inhibitor as substrates. HvTrxh1 and HvTrxh2 were also efficiently reduced by Arabidopsis thaliana NADP-dependent thioredoxin reductase (NTR). The biochemical properties of HvTrxh2 and TrxTa were similar, whereas HvTrxh1 had higher insulin-reducing activity and was a better substrate for Arabidopsis NTR than HvTrxh2, with a Km of 13 micro m compared with 44 micro m for HvTrxh2. Thus, barley seeds contain two distinct thioredoxin h isoforms which differ in temporal and spatial distribution and kinetic properties, suggesting that they may have different physiological roles.
A KCl-soluble, albumin/globulin fraction of wheat (Triticum aestivum L.) starchy endosperm was further separated into a methanol-insoluble fraction that contained metabolic proteins and a methanol-soluble fraction that contained "chloroform-methanol" or CM-like proteins. Reduction of the disulfide bonds of the CM proteins with thioredoxin or dithiothreitol altered their properties so that, like the metabolic proteins, they were insoluble in methanol. Glutathione had little effect, indicating dithiol specificity. Proteomic analysis of the CM protein fraction revealed the presence of isoforms of low molecular weight disulfide proteins (alpha-amylase, alpha-amylase/trypsin and WCI proteinase inhibitors, lipid transfer proteins, gamma-thionins), stress enzymes (Cu-Zn superoxide dismutase and peroxidase), storage proteins (alpha-, gamma- and omega-gliadins, low molecular weight glutenin subunits and globulins of the avenin N9 type), and a component of protein degradation (polyubiquitin). These findings support the view that, in addition to modifying activity and increasing protease sensitivity, reduction by thioredoxin alters protein solubility, thereby promoting processes of the grain starchy endosperm, notably the mobilization of reserves during germination and seedling development.
There is growing interest in the proteins involved in protein folding. This is mainly due to the large number of human diseases related to defects in folding, which include cystic fibrosis, Alzheimer's and cancer. However, equally important as the oxidation and concomitant formation of disulfide bridges of the extracellular or secretory proteins is the reduction and maintenance in the reduced state of the proteins within the cell. Interestingly, the proteins that are responsible for maintenance of the reduced state belong to the same superfamily as those responsible for the formation of disulfide bridges: all are members of the thioredoxin superfamily. In this article, we highlight the main features of those thioredoxin-like proteins directly involved in the redox reactions. We describe their biological functions, cytoplasmic location, mechanisms of action, structures and active site features, and discuss the principal hypotheses concerning origins of the different reduction potentials and unusual pK(a)'s of the catalytic residues.
Thioredoxin is ubiquitous and regulates various target proteins through disulfide bond reduction. We report the structure of thioredoxin (HvTrxh2 from barley) in a reaction intermediate complex with a protein substrate, barley alpha-amylase/subtilisin inhibitor (BASI). The crystal structure of this mixed disulfide shows a conserved hydrophobic motif in thioredoxin interacting with a sequence of residues from BASI through van der Waals contacts and backbone-backbone hydrogen bonds. The observed structural complementarity suggests that the recognition of features around protein disulfides plays a major role in the specificity and protein disulfide reductase activity of thioredoxin. This novel insight into the function of thioredoxin constitutes a basis for comprehensive understanding of its biological role. Moreover, comparison with structurally related proteins shows that thioredoxin shares a mechanism with glutaredoxin and glutathione transferase for correctly positioning substrate cysteine residues at the catalytic groups but possesses a unique structural element that allows recognition of protein disulfides.
DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of a periplasmic, N-terminal domain (nDsbD), a central transmembrane domain and a periplasmic, C-terminal domain (cDsbD). Each of these domains contains two essential cysteine residues that are required for intermolecular disulfide exchange between DsbD and substrates, and intramolecular disulfide exchange between the three DsbD domains. In order to determine the rate of intramolecular electron transfer from cDsbD to nDsbD, we constructed a redox-sensitive tryptophan variant of cDsbD (cDsbD(W)) that shows an approximately threefold increase in fluorescence upon reduction and has the same redox potential and reactivity as wild-type cDsbD. cDsbD(W) was then used for the construction of fusion proteins with nDsbD and cDsbD(W), connected via flexible linkers of different length. Using the DsbD substrate DsbC, which can only be reduced by nDsbD and does not react with cDsbD, we could directly measure the intramolecular electron transfer from cDsnD(W) to nDsbB in the fusion proteins. We show that the intramolecular disulfide exchange is significantly faster than the reaction between isolated nDsbD and cDsbD. Nevertheless, the effective concentration of 0.2 mM of the domains in the fusions is comaparably low. The rate of 23 s(-1) for the intramolecular disulfide exchange in the fusions was independent of the linker length and may represent the upper limit for the substrate turnover of full-length DsbD.
Neuronal calcium sensor-1 (NCS-1) is a major modulator of Ca(2+) signaling with a known role in neurotransmitter release. NCS-1 has one cryptic (EF1) and three functional (EF2, EF3, and EF4) EF-hand motifs. However, it is not known which are the regulatory (Ca(2+)-specific) and structural (Ca(2+)- or Mg(2+)-binding) EF-hand motifs. To understand the specialized functions of NCS-1, identification of the ionic discrimination of the EF-hand sites is important. In this work, we determined the specificity of Ca(2+) binding using NMR and EF-hand mutants. Ca(2+) titration, as monitored by [(15)N,(1)H] heteronuclear single quantum coherence, suggests that Ca(2+) binds to the EF2 and EF3 almost simultaneously, followed by EF4. Our NMR data suggest that Mg(2+) binds to EF2 and EF3, thereby classifying them as structural sites, whereas EF4 is a Ca(2+)-specific or regulatory site. This was further corroborated using an EF2/EF3-disabled mutant, which binds only Ca(2+) and not Mg(2+). Ca(2+) binding induces conformational rearrangements in the protein by reversing Mg(2+)-induced changes in Trp fluorescence and surface hydrophobicity. In a larger physiological perspective, exchanging or replacing Mg(2+) with Ca(2+) reduces the Ca(2+)-binding affinity of NCS-1 from 90 nM to 440 nM, which would be advantageous to the molecule by facilitating reversibility to the Ca(2+)-free state. Although the equilibrium unfolding transitions of apo-NCS-1 and Mg(2+)-bound NCS-1 are similar, the early unfolding transitions of Ca(2+)-bound NCS-1 are partially influenced in the presence of Mg(2+). This study demonstrates the importance of Mg(2+) as a modulator of calcium homeostasis and active-state behavior of NCS-1.
H-type thioredoxins (Trxs) constitute a particularly large Trx sub-group in higher plants. Here, the crystal structures are determined for the two barley Trx h isoforms, HvTrxh1 and HvTrxh2, in the partially radiation-reduced state to resolutions of 1.7 A, and for HvTrxh2 in the oxidized state to 2.0 A. The two Trxs have a sequence identity of 51% and highly similar fold and active-site architecture. Interestingly, the four independent molecules in the crystals of HvTrxh1 form two relatively large and essentially identical protein-protein interfaces. In each interface, a loop segment of one HvTrxh1 molecule is positioned along a shallow hydrophobic groove at the primary nucleophile Cys40 of another HvTrxh1 molecule. The association mode can serve as a model for the target protein recognition by Trx, as it brings the Met82 Cgamma atom (gamma position as a disulfide sulfur) of the bound loop segment in the proximity of the Cys40 thiol. The interaction involves three characteristic backbone-backbone hydrogen bonds in an antiparallel beta-sheet-like arrangement, similar to the arrangement observed in the structure of an engineered, covalently bound complex between Trx and a substrate protein, as reported by Maeda et al. in an earlier paper. The occurrence of an intermolecular salt bridge between Glu80 of the bound loop segment and Arg101 near the hydrophobic groove suggests that charge complementarity plays a role in the specificity of Trx. In HvTrxh2, isoleucine corresponds to this arginine, which emphasizes the potential for specificity differences between the coexisting barley Trx isoforms.
Protein–protein interactionsinplant
- Y Meyer
- Miginiac
- M Maslow
- P Schürmann
- J P Jacquot
- M T Mcmanus
- W Laing
- A Allan
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Protein-protein interactions in plant thioredoxin dependent systems
- Y Meyer
- M Miginiac-Maslow
- P Schürmann
- J P Jacquot
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- M Miginiac-Maslow
- S K Kim
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- J P Jacquot
- C C Longbine
- F De Lamotte-Guery
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Maslow, M., Kim, S.K., Mason, J., Jacquot, J.P., Longbine, C.C., de Lamotte-Guery,
F. and Knaff, D.B. (2003) Effect of pH on the oxidation–reduction properties of
thioredoxins. Biochemistry 42, 14877–14884.
Physiological functions of thioredoxin and thioredoxin reductase
- E S J Arnér
- A Holmgren
Arnér, E.S.J. and Holmgren, A. (2000) Physiological functions of thioredoxin
and thioredoxin reductase. Eur. J. Biochem. 267, 6102-6109.
Thioredoxin reduction alters the solubility of proteins of wheat
- J H Wong
- N Cai
- C K Tanaka
- W H Vensel
- W J Hurkman
- B B Buchanan
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B.B. (2004) Thioredoxin reduction alters the solubility of proteins of wheat