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PubMed2004

Role of disulfide bonds for the structure and folding of proguanylin.

The intestinal peptide hormone guanylin circulates mainly as its corresponding prohormone of 94 amino acids and is the first identified endogenous ligand of intestinal guanylyl cyclase C. While the prohormone is biologically inactive, it is processed to the fully functional form with 15 amino acid residues corresponding to the COOH terminus of the precursor protein. In addition to this inactivation of the hormone region, the prosequence makes an essential contribution to the disulfide-coupled folding of the hormone. On the basis of the recently determined solution structure of proguanylin, explanations for these functions of the prosequence were found, indicating that interstrand contacts between the NH2-terminal beta-hairpin of the prosequence and the COOH-terminal hormone region are crucial for formation of the correct disulfide bonds of guanylin. To further investigate the role of individual disulfide bonds upon stabilization of the overall three-dimensional structure of proguanylin and to test the assumption of a direct effect of the prosequence on the structure of the hormone region, we studied the cysteine double mutant proteins proguanylin-C48S/C61S and proguanylin-C86S/C94S. Disulfide determination as well as CD and NMR spectroscopy of proguanylin-C48S/C61S reveals an essential function of the Cys48-Cys61 disulfide bond for the stability of the hydrophobic core and thereby for the stability of the overall three-dimensional structure of proguanylin. Furthermore, sequence specific resonance assignment of the second disulfide deletion mutant, proguanylin-C86S/C94S, and comparison of the NMR spectra of this protein with those of the wild-type protein demonstrate that the rigid helical core structure of proguanylin is not affected by the mutation. Additionally, analysis of the interstrand contacts between the termini reveals a direct effect of the prosequence on the conformation of the hormone region. On the basis of these results, we propose a cooperative mechanism that leads to formation of the correct disulfide pattern of guanylin.

Amino Acid Sequence↗
PubMed2005

Increasing the reactivity of an artificial dithiol-disulfide pair through modification of the electrostatic milieu.

The thiol-disulfide exchange reaction plays a central role in the formation of disulfide bonds in newly synthesized proteins and is involved in many aspects of cellular metabolism. Because the thiolate form of the cysteine residue is the key reactive species, its electrostatic milieu is thought to play a key role in determining the rates of thiol disulfide exchange reactions. While modest reactivity effects have previously been seen in peptide model studies, here, we show that introduction of positive charges can have dramatic effects on disulfide bond formation on a structurally restricted surface. We have studied properties of vicinal cysteine residues in proteins using a model system based on redox-sensitive yellow fluorescent protein (rxYFP). In this system, the formation of a disulfide bond between two cysteines Cys149 and Cys202 is accompanied by a 2.2-fold decrease in fluorescence. Introduction of positively charged amino acids in the proximity of the two cysteines resulted in an up to 13-fold increase in reactivity toward glutathione disulfide. Determination of the individual pK(a) values of the cysteines showed that the observed increase in reactivity was caused by a decrease in the pK(a) value of Cys149, as well as favorable electrostatic interactions with the negatively charged reagents. The results presented here show that the electrostatic milieu of cysteine thiols in proteins can have substantial effects on the rates of the thiol-disulfide exchange reactions.

Bacterial Proteins↗
PubMed2005

Cys-10 mixed disulfide modifications exacerbate transthyretin familial variant amyloidogenicity: a likely explanation for variable clinical expression of amyloidosis and the lack of pathology in C10S/V30M transgenic mice?

The marked variation in clinical expression and age of familial amyloid disease onset is not well understood. One possibility is that metabolite modification(s) of a disease-associated mutant protein can change the energetics and propensity for misfolding, influencing the disease course. Each subunit of the transthyretin (TTR) tetramer has a single Cys residue that can exist in the SH form or as a mixed disulfide with the amino acid Cys or the peptide glutathione or fragments of the latter. The stability and amyloidogenicity of the clinically most important TTR variants (V30M and V122I) in their SH oxidation state were compared with those of their mixed disulfide adducts. All the Cys-10 mixed disulfide conjugates exhibited substantially decreased protein stability (urea, pH 7) and a higher rate and extent of amyloidogenesis (slightly acidic conditions). We also investigated the amyloidogenicity and stability of a C10S/V30M TTR double mutant which lacks the ability to make mixed disulfides, but retains the disease-associated V30M mutation. Unlike V30M TTR, this double mutant is nonamyloidogenic in transgenic mice. Our in vitro data reveal that the C10S/V30M and V30M TTR homotetramers have identical amyloidogenicity and stability, implying that Cys-10 mixed disulfide formation enhances amyloidogenesis in V30M transgenic mice. Given the high proportion of TTR subunits having mixed disulfide modifications in human plasma ( approximately 50%), and the data within demonstrating their increased amyloidogenicity, we submit that disulfide metabolite modifications have the potential to influence the course of amyloidoses, including TTR amyloidoses caused by mutations.

Amyloid↗
PubMed2006

Pathway of oxidative folding of secretory leucocyte protease inhibitor: an 8-disulfide protein exhibits a unique mechanism of folding.

Secretory leucocyte protease inhibitor (SLPI) is a 107-amino acid protein with a high density of disulfide pairing (eight). The mechanism of oxidative folding of reduced and denatured SLPI has been investigated here. Despite an exceedingly large number of possible folding intermediates ( approximately 46 million disulfide isomers) and their potential to complicate the refolding process, oxidative folding of SLPI turns out to be surprisingly simple and efficient. Complete oxidative folding and a near-quantitative recovery of the native SLPI can be achieved in a simple buffer solution using air oxidation without any supplementing thiol catalyst or redox agent, a phenomenon that has not yet been observed with other disulfide proteins. Because of the heterogeneity and extensive overlapping of folding intermediates, identification of the predominant intermediate was unfeasible. Nonetheless, studies of reductive unfolding of native SLPI and oxidative folding of a six-disulfide variant of SLPI enable us to propose an underlying mechanism accounting for the unique folding efficiency of SLPI in the absence of a redox agent. Our studies indicate that oxidative folding of SLPI undergoes heterogeneous populations of one-, two-, three-, four-, five-, six-, and seven-disulfide isomers, including two nativelike isomers, SLPI-6A and SLPI-7A, as transient intermediates. Formation of the last two native disulfide bonds leading to the conversion of SLPI-6A --> SLPI-7A --> N-SLPI is relatively slow and represents the final stage of oxidative folding. Most importantly, free cysteines of SLPI-6A and SLPI-7A also act as a thiol catalyst in promoting the disulfide shuffling of diverse non-native intermediates accumulated along the folding pathway. This explains why a near-quantitative recovery of N-SLPI can be achieved in the absence of any thiol catalyst and redox agent. Properties of SLPI-6A and SLPI-7A were investigated and compared to those of other documented kinetic intermediates of oxidative folding. The correlation between the mechanism of SLPI folding and the three-dimensional structure of SLPI is also elaborated.

Amino Acid Sequence↗
PubMed2006

The location of an engineered inter-subunit disulfide bond in factor for inversion stimulation (FIS) affects the denaturation pathway and cooperativity.

Two crossed-linked variants of the homodimeric DNA binding protein factor for inversion stimulation (FIS) were created via engineering of single intermolecular disulfide bonds. The conservative S30C and the nonconservative V58C FIS independent mutations resulted in FIS crossed-linked at the A helix (C30-C30) and at the middle of the B helix (C58-C58). This study sought to investigate how the location of an intermolecular disulfide bond may determine the effect on stability and its propagation through the structure to preserve or alter the denaturation cooperativity of FIS. The oxidized and reduced S30C and V58C FIS exhibited a far-UV CD spectrum and DNA binding affinities that were similar to WT FIS, indicating no significant changes in secondary and tertiary structure. However, the reduced and oxidized forms of the mutants revealed significant differences in the stability and equilibrium denaturation mechanism between the two mutants. In the reduced state, S30C FIS had very little effect on FIS stability, whereas V58C FIS was 2-3 kcal/mol less stable than WT FIS. Interestingly, while both disulfide bonds significantly increased the resistance to urea- and guanidine hydrochloride (GuHCl)-induced denaturation, oxidized V58C FIS exhibited a three-state GuHCl-induced transition. In contrast, oxidized S30C FIS displayed a highly cooperative WT-like transition with both denaturants. The three-state denaturation mechanism of oxidized V58C FIS induced by the GuHCl salt was reproduced by urea denaturation at pH 4, suggesting that disruption of a C-terminus salt-bridge network is responsible for the loss of denaturation cooperativity of V58C FIS in GuHCl or urea, pH 4. A second mutation on V58C FIS created to place a single tryptophan probe (Y95W) at the C-terminus further implies that the denaturation intermediate observed in disulfide crossed-linked V58C FIS results from a decoupling of the stabilities of the C-terminus and the rest of the protein. These results show that, unlike the C30-C30 intermolecular disulfide bond, the C58-C58 disulfide bond did not evenly stabilize the FIS structure, thereby highlighting the importance of the location of an engineered disulfide bond on the propagation of stability and the denaturation cooperativity of a protein.

Circular Dichroism↗
PubMed1996

Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation.

A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.

Amino Acid Sequence↗
PubMed1997

"Designing out" disulfide bonds: thermodynamic properties of 30-51 cystine substitution mutants of bovine pancreatic trypsin inhibitor.

We have used a combination of spectroscopic and calorimetric techniques to assess the thermodynamic and extrathermodynamic consequences of paired amino acid substitutions at positions 30 and 51 in bovine pancreatic trypsin inhibitor (BPTI). Correctly folded, wild type BPTI contains a disulfide at the 30-51 positions, with the nonbackbone atoms of this cystine being relatively solvent inaccessible. Mutants missing this buried 30-51 disulfide adopt a conformation very similar to that of the native state of wild type BPTI (Eigenbrot et al., 1990, 1992), although they are severely destabilized relative to the wild type molecule (Hurle et al., 1990). We have conducted a systematic effort to find the energetically most favorable substitution for this buried 30-51 disulfide in BPTI. To this end, we have studied and characterized the thermally induced and guanidine hydrochloride-induced denaturation transitions for a family of mutants in which the amino acid residue(s) at positions 30 and/or 51 have been systematically altered. Specifically, we studied the unfolding transitions of the following series of residue 30/residue 51 paired substitution mutants: C30A/C51A, C30V/C51A, C30G/C51A, C30S/C51A, C30T/C51A, C30A/C51S, C30S/C51S, and C30G/C51M. For this series of mutants, comparisons between the relative stabilization free energies, derived from analysis of the denaturation profiles, allow us to reach the following conclusions: (a) side chains containing polar moieties (Ser and Thr) are destabilizing, with this effect being position dependent (i.e., a serine substitution is more destabilizing at position 51 than at position 30); (b) the destabilizing effects of two serine substitutions are approximately additive, suggesting that side chain-side chain hydrogen bonds between the two serine hydroxyl groups probably are weak or nonexistent; (c) the thermodynamic impact of a Gly30 substitution is consistent with a glycine-induced increase in the configurational entropy of the unfolded state; (d) the C30G/C51M mutant is highly destabilized relative to the C30A/C51A mutant despite the fact that, based on considerations of hydrophobicity and steric fit, substitution of a buried disulfide by Gly30 and Met51 would be expected to be optimal. Methionine may be particularly ill-suited as a buried disulfide substitute due to the large loss of side chain conformational entropy it undergoes during the transition from the unfolded to the native state. In the aggregate, our data provide insight into the residue-, position-, and context-dependent consequences on protein stability of "designing out" the buried 30-51 disulfide bond in the BPTI molecule. These data also suggest that a previously unrecognized component of disulfide bridge stabilization of proteins is the relatively minor penalty in side chain conformational entropy incurred by cystine residues during folding due to their severely restricted rotation even in the unfolded state.

Amino Acid Sequence↗
PubMed1998

Formation of native disulfide bonds in endothelin-1. Structural evidence for the involvement of a highly specific salt bridge between the prosequence and the endothelin-1 sequence.

The [Lys-Arg]-endothelin-1 analogue (KR-ET-1) yields almost selectively the native disulfide pattern (96%), in contrast to endothelin-1 (ET-1) that gives at least 25% of the non-native disulfide pattern. We have previously shown that the carboxylate-state structure of KR-ET-1 is more constrained and stabilized by a salt bridge between Arg(-1) and the Asp8 or Glu10 side chain [Aumelas et al. (1995) Biochemistry 34, 4546-4561]. To identify this salt bridge and its potential involvement in the disulfide bond formation, [E10Q], [D18N], and [D8N] carboxamide analogues were studied, which led to the unambiguous identification of the Arg(-1)-Asp8 salt bridge. Furthermore, while [E10Q] and [D18N] analogues gave a high yield of the native isomer (>/=90%), the [D8N] analogue afforded a ratio of the two isomers close to that observed for ET-1 (68%) [Kubo et al. (1997) Lett. Pept. Sci. 4, 185-192]. Assuming that the formation of disulfide bonds occurs in a thermodynamically controlled step, we have hypothesized that the Arg(-1)-Asp8 salt bridge and concomitant interactions could be responsible for the increase in yield of the native isomer of KR-ET-1. In the present work, we describe the structural studies of the carboxamide analogues and of the minor non-native KR-ET-1 isomer. On the basis of 1H NMR and CD spectra as a function of pH, [E10Q] and [D18N] analogues display a conformational change similar to that of the parent peptide, whereas the structure of the [D8N] analogue is unchanged. For the non-native isomer, we measured a lower helical content than for the native isomer and observed a marked difference in the orientation of the KRCSC backbone. In addition, no salt bridge was experimentally observed. Altogether, these results allow us to hypothesize that the salt bridge between two highly conserved residues, one belonging to the prosequence [Arg(-1)] and the other to the mature sequence [Asp8], is involved in the formation of the native disulfide isomer of ET-1. The involvement of the prosequence in the formation of the native disulfide isomer strongly suggests that, in the maturation pathway of ET-1, cleavage of the Arg52-Cys53 amide bond occurs after native disulfide bond formation.

Amino Acid Sequence↗
PubMed1998

In vitro disulfide-coupled folding of guanylyl cyclase-activating peptide and its precursor protein.

Guanylyl cyclase-activating peptide II (GCAP-II), an endogenous ligand of particulate guanylyl cyclase C (GC-C), is processed from the precursor protein and circulates in human blood. GCAP-II consists of 24 amino acid residues and contains two disulfide bridges. The correct disulfide paring of GCAP-II is an absolute requirement for its biological activity. This study shows that the folding of the peptide from the reduced form yields a peptide with the native disulfide paring as a minor product and with non-native ones as major products, regardless of the presence or absence of reduced and oxidized glutathione. The results suggest that GCAP-II does not possess sufficient information to permit the adoption of the native conformation and to effectively form the correct disulfide pairing and, as a result, that GCAP-II is correctly folded by assistance of a factor(s) such as an intra- or intermolecular chaperone. We studied whether a peptide in the pro-leader sequence of the precursor protein (proGCAP-II) contains sufficient information to facilitate the folding of GCAP-II. For this purpose, we prepared proGCAP-II in Escherichia coli by a recombinant technique and examined the disulfide-coupled folding of proGCAP-II from the reduced form. proGCAP-II was quantitatively recovered with the correctly folded structure from the reduced form both in the presence and in the absence of reduced and oxidized glutathione. The protein contains only disulfide linkages at the same positions as the mature form of proGCAP-II, GCAP-II, and the biologically active isomer of GCAP-II in the molecule. These results provide evidence that the propeptide of proGCAP-II is a critical factor in the formation of the correct disulfide paring in the folding of the protein.

Amino Acid Sequence↗
PubMed1999

Role of individual cysteine residues and disulfide bonds in the structure and function of Aspergillus ribonucleolytic toxin restrictocin.

Restrictocin, produced by the fungus Aspergillus restrictus, belongs to the group of ribonucleolytic toxins called ribotoxins. It specifically cleaves a single phosphodiester bond in a conserved stem and loop structure in the 28S rRNA of large ribosomal subunit and potently inhibits eukaryotic protein synthesis. Restrictocin contains 149 amino acid residues and includes four cysteines at positions 5, 75, 131, and 147. These cysteine residues are involved in the formation of two disulfide bonds, one between Cys 5 and Cys 147 and another between Cys 75 and Cys 131. In the current study, all four cysteine residues were changed to alanine individually and in different combinations by site-directed mutagenesis so as to remove one or both the disulfides. The mutants were expressed and purified from Escherichia coli. Removal of any cysteine or any one of the disulfide bonds individually did not affect the ability of the toxin to specifically cleave the 28S rRNA or to inhibit protein synthesis in vitro. However, the toxin without both disulfide bonds completely lost both ribonucleolytic and protein synthesis inhibition activities. The active mutants, containing only one disulfide bond, exhibited relatively high susceptibility to trypsin digestion. Thus, none of the four cysteine residues is directly involved in restrictocin catalysis; however, the presence of any one of the two disulfide bonds is absolutely essential and sufficient to maintain the enzymatically active conformation of restrictocin. For maintenance of the unique stability displayed by the native toxin, both disulfide bonds are required.

Allergens↗
PubMed1999

Role of disulfide bonds in the structure and potassium channel blocking activity of ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35 residue polypeptide cross-linked by three disulfide bridges: Cys3-Cys35, Cys12-Cys28, and Cys17-Cys32. To investigate the role of these disulfides in the structure and channel-blocking activity of ShK toxin, a series of analogues was synthesized by selective replacement of each pair of half-cystines with two alpha-amino-butyrate (Abu) residues. The remaining two disulfide pairs were formed unambiguously using an orthogonal protecting group strategy of Cys(Trt) or Cys(Acm) at the appropriate position. The peptides were tested in vitro for their ability to block Kv1.1 and Kv1.3 potassium channels and their ability to displace [(125)I]dendrotoxin binding to rat brain synaptosomal membranes. The monocyclic peptides showed no activity in these assays. Of the dicyclic peptides, [Abu12,28]ShK(3-35,17)(-)(32) (where the subscript indicates disulfide connectivities) had weak activity on Kv1.3 and Kv1.1. [Abu17,32]ShK(3-35,12)(-)(28) blocked Kv1.3 with low nanomolar potency, but was less effective (being comparable to [Abu12,28]ShK(3-35,17)(-)(32)) against Kv1.1. [Abu3, 35]ShK(12-28,17)(-)(32), retained high picomolar affinity against both channels. Corroborating these results, [Abu3,35]ShK(12-28, 17)(-)(32) had an IC(50) ratio relative to native toxin of 18 in the displacement assay, whereas [Abu17,32]ShK(3-35,12)(-)(28) and [Abu12, 28]ShK(3-35,17)(-)(32) had ratios of 69 and 390, respectively. Thus, the disulfide bond linking the N- and C-terminal regions is less important for activity than the internal disulfides. NMR analysis of the [Abu12,28] and [Abu17,32] analogues indicated that they had little residual structure, consistent with their significantly reduced activities. By contrast, [Abu3,35]ShK(12-28,17)(-)(32) had a moderately well-defined solution structure, with a mean pairwise root-mean-square deviation of 1.33 A over the backbone heavy atoms. This structure nevertheless showed significant differences from that of native ShK toxin. The possible interactions of this analogue with the channel and the distinction between native secondary and tertiary structure on one hand and global topology imposed by the disulfide bridges on the other are discussed.

Amino Acid Sequence↗
PubMed2000

Disulfide bridge engineering in the tachykinin NK1 receptor.

As in most other seven-transmembrane receptors, the central disulfide bridge from the extracellular end of TM-III to the middle of the second extracellular loop was essential for ligand binding in the NK1 receptor. However, introduction of "extra", single Cys residues in the second extracellular loop, at positions where disease-associated Cys substitutions impair receptor function in the vasopressin V2 receptor and in rhodopsin, did not cause mispairing with the Cys residues involved in this central disulfide bridge. Cys residues were introduced in the N-terminal extension and in the third extracellular loop, respectively, in such a way that disulfide bridge formation could be monitored by loss of substance P binding and breakage of the bridge could be monitored by gain of ligand binding. This disulfide bridge formed spontaneously in the whole population of receptors and could be titrated with low concentrations of reducing agent, dithiothreitol. Another putative disulfide bridge "switch" was constructed at the extracellular ends of TM-V and -VI, i.e., at positions where a high-affinity zinc site previously had been constructed with His substitutions. Disulfide bridge formation at this position, monitored by loss of binding of the nonpeptide antagonist [3H]LY303.870, occurred spontaneously only in a small fraction of the receptors. It is concluded that disulfide bridges form readily between Cys residues introduced appropriately in the N-terminal extension and the third extracellular loop, whereas they form with more difficulty between Cys residues placed at the extracellular ends of the transmembrane segments even at positions where high-affinity metal ion sites can be constructed with His residues.

Amino Acid Sequence↗
PubMed2003

Assessing the magnitude of folding forces along the oxidative folding pathway of multi-disulfide-containing proteins.

Oxidative folding involves the acquisition of the native set of disulfide bonds and native structure of a protein from its reduced, unfolded form. Two major factors that influence the distribution of disulfide bonds (over their entropically favored probability of formation) during the regeneration, eventually favoring and sustaining the native set of disulfide bonds, are enthalpic interactions that lead to conformational ordering among intermediates and conformational folding, resulting in native (or nativelike) structure which protects the native disulfide bonds from reduction and intramolecular thiol-disulfide exchange reactions. Here, we report a novel technique to provide a quantitative description of the enthalpic and conformational folding forces present at any stage of the oxidative folding process of multi-disulfide-containing proteins which is made feasible by assessing their ability to form "structures" that are able to protect disulfide bonds from reduction.

Animals↗
PubMed1999

Controlled syntheses of natural and disulfide-mispaired regioisomers of alpha-conotoxin SI.

Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of alpha-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two 'interlocking' disulfide bridges connecting Cys2-Cys7 and Cys3-Cys13 (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys2-Cys13 and Cys3-Cys7 (2/13 & 3/7, 'nested') and Cys2-Cys3 and Cys7-Cys13 (2/3 & 7/13, 'discrete'), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation of S-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.

Amino Acid Sequence↗
PubMed2000

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ¿i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.

Amino Acid Sequence↗
PubMed2004

Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS.

The antioxidant enzyme Cu,Zn-superoxide dismutase (SOD1) has the distinction of being one of the most abundant disulfide-containing protein known in the eukaryotic cytosol; however, neither catalytic nor physiological roles for the conserved disulfide are known. Here we show that the disulfide status of Saccharomyces cerevisiae SOD1 significantly affects the monomer-dimer equilibrium, the interaction with the copper chaperone CCS, and the activity of the enzyme itself. Disulfide formation in SOD1 by O2 is slow but is greatly accelerated by the Cu-bound form of CCS (Cu-CCS) in vivo and in vitro even in the presence of excess reductants; once formed, this disulfide is kinetically stable. Biochemical assays reveal that Cu-CCS facilitates Cys oxidation and disulfide isomerization in the stepwise conversion of the immature form of the enzyme to the active state. The immature form of SOD1 is most susceptible to oxidative insult and to aggregation reminiscent of that observed in amyotrophic lateral sclerosis. Thus Cu-CCS mediation of correct disulfide formation in SOD1 is important for regulation of enzyme activity and for prevention of misfolding or aggregation.

Binding Sites↗
PubMed2001

The multidrug resistance protein MRP1 mediates the release of glutathione disulfide from rat astrocytes during oxidative stress.

The release of glutathione disulfide has been considered an important process for the maintenance of a reduced thiol redox potential in cells during oxidative stress. In cultured rat astrocytes, permanent hydrogen peroxide-induced oxidative stress caused a rapid increase in intracellular glutathione disulfide, which was followed by the appearance of glutathione disulfide in the medium. Under these conditions, the viability of the cells was not compromised. In the presence of cyclosporin A and the quinoline-derivative MK571, inhibitors of multidrug resistance proteins (MRP1 and MRP2), glutathione disulfide accumulated in cells and the release of glutathione disulfide from astrocytes during H2O2 stress was potently inhibited, suggesting a contribution of MRP1 or MRP2 in the release of glutathione disulfide from astrocytes. Using RT-PCR we amplified a cDNA from astroglial RNA with a high degree of homology to MRP1 from humans and mouse. In contrast, no fragment was amplified by using primers specific for rat MRP2. In addition, the presence of MRP1 protein in astrocytes was demonstrated by its immunolocalization in cells expressing the astroglial marker protein glial fibrillary acidic protein. Our data identify rat astrocytes as a MRP1-expressin, brain cell type and demonstrate that this transporter participates in the release of glutathione disulfide from astrocytes during oxidative stress.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1981

Subunit structure of cell surface proteins: disulfide bonding in antigen receptors, Ly-2/3 antigens, and transferrin receptors of murine T and B lymphocytes.

The surface proteins of lymphocytes from spleen and thymus and several cultured lymphoid tumor lines were radioiodinated in situ, solubilized with Triton X-100, and examined for the presence of disulfide-bonded subunits by two-dimensional (intact, reduced) NaDodSO4/polyacrylamide gel electrophoresis. [Hynes, R. O. & Destree, A. (1977) Proc. Natl. Acad. Sci. USA 74, 2855-2859]. Few lymphocyte surface proteins were found to consist of disulfide-bonded subunits, and the most prominent of these could be identified. In normal B lymphocytes and B-lymphoma cells, IgD or IgM (or both) were the major disulfide-bonded proteins, and these were easily detectable, even without immunoprecipitation. In contrast, analysis of thymocytes and T-lymphoma cells did not reveal any protein resembling immunoglobulin in its chain structure. The major labeled thymocyte membrane protein consisting of disulfide-bonded subunits was identified as the Ly-2/3 antigen. It appeared to contain disulfide-bonded homodimers of Mr 35,000 (alpha 2) noncovalently associated with a second pair of homodimers of Mr 30,000 (beta 2). Peptide mapping showed these polypeptides to be homologous. A third disulfide-bonded homodimer, which was heterogeneous in apparent Mr, appeared to be part of the Ly-2/3 complex. All cultured T- and B-lymphoma lines examined were found to possess a major surface protein that appeared to be a disulfide-bonded homodimer of a polypeptide of Mr 95,000. This protein was identified as the receptor for transferrin. It is suggested that the presence of two or more subunits in cell surface receptors renders their ligand functionally bivalent, making ligand-induced receptor aggregation possible.

Animals↗