Analysis of IL-2 functional structure by multiple cysteine substitutions.

IL-2 has three cysteine residues. The cysteines at positions 58 and 105 of active IL-2 form an intramolecular disulfide bond while that at position 125 remains as a free form. To evaluate the importance of correct disulfide bond, mutant proteins (muteins) that have triple and double substitutions of cysteines with alanines, namely A58/105/125 and A58/125, were made by polymerase chain reaction method respectively. Thymidine incorporation assay on CTLL-2 cells showed that although these two muteins were only 0.5-2.0% as potent as that of wild type IL-2, they were 50-200 fold more active than A58, a mutein that has substitution of cysteine at position 58 with alanine. Binding inhibition study showed that the relative affinity of muteins A58/125 and A58/105/125 for high affinity IL-2 receptors was 5-25 fold higher than that of A58. These results suggest that the dramatic decrease in the activity of mutein A58 may result from the formation of an incorrect disulfide bond between the cysteines at positions 105 and 125.     

Importance of disulfide linkage for constructing the biologically active human interleukin-2

Recombinant human interleukin-2 (rIL-2) produced in Escherichia coli possesses a free thiol group at Cys-125 and a disulfide linkage between Cys-58 and Cys-105, as in the case for natural human interleukin-2. Treatment of rIL-2 with 200 mM dithiothreitol resulted in the cleavage of the Cys-58-Cys-105 disulfide bond. The reduced form of rIL-2 thus obtained retained only 10% of the in vitro biological activity of the native form, as measured by the ability to stimulate the growth of an IL-2-dependent mouse natural killer cell line, NKC3. Far-uv circular dichroism studies indicated that the cleavage of the disulfide bond results in a decrease of alpha-helix content. Near-uv circular dichroism studies suggested that the native molecule is folded into a rigid tertiary structure, while the reduced form showed a spectrum similar to that of rIL-2 denatured in the presence of 6 M guanidine.HCl. The once-reduced molecule was readily reoxidized in the presence of 10 microM Cu2+ to form the native molecule with full biological activity. These results strongly demonstrate that the Cys-58-Cys-105 disulfide linkage in the IL-2 molecule is essential for constructing a rigid and biologically active form of IL-2.     

Biological characterization of human interleukin-2 mutant proteins. Structure-activity relationship studies

Several human interleukin-2 (IL-2) mutant proteins have been produced previously by site-directed mutagenesis and found to have different capacities to induce T-cell proliferative activity. In this study, the abilities of these IL-2 mutant proteins to activate natural killer cells and to induce interferon-gamma production have been evaluated, and the binding of these proteins to IL-2 receptors analyzed. Natural killer cell activation and interferon-gamma induction assays showed that the relative activities of IL-2 mutant proteins were consistent with their relative activities in T-cell proliferation assay. Receptor-binding studies showed that the activities of most proteins correlated well with their respective affinities for high-affinity IL-2 receptors on CTLL-2 cells. Interestingly, although the mutant protein with deletion of cysteine 125 (des-Cys125) was biologically less active than the protein with substitution of alanine for cysteine 105 (Ala105), both proteins exhibited similar affinity. Des-Cys125, like IL-2 and Ala105, also caused down-regulation of high-affinity IL-2 receptors. Binding studies on MLA-144, a cell line expressing mainly intermediate-affinity IL-2 receptors (IL-2R beta), however, showed that des-Cys125 had much lower affinity than Ala105. These results suggest that binding of IL-2 and mutant proteins to the IL-2R beta component of the high-affinity receptor is essential for the induction of biological effects.     

Disulfide scrambling of interleukin-2: HPLC resolution of the three possible isomers

Native interleukin-2 (IL-2) contains three cysteines; two exist in a disulfide bridge (Cys-58 and Cys-105) and the third Cys-125 is a free sulfhydryl. In the presence of 6 M guanidine hydrochloride at alkaline pH, IL-2 is converted into three isomers. Each isomer represents one of the three possible disulfide-linked forms that can be generated from three cysteines. These three isomers were resolved on a C4 reverse-phase HPLC system. The identity of each of the three forms was determined by carboxymethylation of the free cysteines in each isomer with [3H]iodoacetic acid followed by determination of the labelled cysteines by tryptic peptide mapping. Tryptic peptide mapping of the more predominant of the two scrambled peaks showed it to be the Cys-105-S-S-Cys-125 linked form of IL-2. A Ser-125 construction of IL-2, which lacks a free cysteine, did not scramble under these conditions. These experiments demonstrate the utility of reverse-phase HPLC in studies of protein folding and disulfide bond structure.     

Cysteine Residues and the Structure of the Rat Renal Proximal Tubular Type II Sodium Phosphate Cotransporter (Rat NaPi IIa)

The rat renal Na/P _i cotransporter type IIa (rat NaP_i IIa) is a 637 amino acid protein containing 12 cysteine residues. We examined the effect of different cysteine modifying methanethiosulfonate (MTS)-reagents and the disulfide bond reducing agent tris(2-carboxyethyl)phosphine (TCEP) on the transport activity of wild-type and 12 single cysteine substitution mutants of rat NaPi IIa expressed in Xenopus laevis oocytes. The transport activity of the wild-type protein was resistant to three membrane impermeant MTS-reagents (MTSEA, MTSET and MTSES). In contrast, membrane permeant methyl methanethiosulfonate (MMTS) and TCEP inhibited the transport activity of both the wild-type, as well as all the single mutant proteins. This indicated the existence of more than one functionally important cysteine residue, not accessible extracellularly, and at least 2 disulfide bridges. To identify the disulfide bridges, three double mutants lacking 2 of the 3 cysteine residues predicted to be extracellular in different combinations were examined. This led to the identification of one disulfide bridge between C306 and C334; reconsideration of the topological model predictions suggested a second disulfide bridge between C225 and C520. Evaluation of a fourth double mutant indicated that at least one of two disulfide bridges (C306 and C334; C225 and C520) has to be formed to allow the surface expression of a functional cotransporter. A revised secondary structure is proposed which includes two partially repeated motifs that are connected by disulfide bridges formed between cysteine pairs C306-C334 and C225-C520. Received: 13 December 1999/Revised: 31 March 2000     

Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding

Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies. Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105. Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies. These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography. Human IL-2 produced in inclusion bodies in E. coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds. Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively. Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.     

Introduction of a non-native disulfide bridge to human lysozyme by cysteine scanning mutagenesis

To examine whether the disulfide bridge between residues 65 and 81 can be replaced by a non-native disulfide bridge in the mutant h-lysozyme C77/95A and whether the formation of such a new disulfide bridge affects the folding of the protein, cysteine scanning mutagenesis has been performed within two discontinuous segments (residues 61-67 for the mutant C65/77/95A, and 74-84 for the mutant C77/81/95A). The position of the Cys residue at 65 or 81 was continuously shifted by site-directed mutagenesis. Of the mutants, only substitution of Cys for Trp64 allowed the secretion of mutant h-lysozyme(W64C) into the medium in a sufficient amount for analysis. After the purification, the mutant enzyme was obtained as two components (W64C-A and W64C-B). The only difference between A and B was that A had a peptide bond cleaved between Ala77 and His78. A non-native disulfide bridge between residues 64-81 was found in both components. Little difference was observed in CD spectra among wild-type and mutant enzymes. It is likely that the tertiary structure of the W64C mutant might be distorted at the location, because the directions of amino acid side chains at positions of 64 and 81 are shown to be opposite to each other in wild-type h-lysozyme by X-ray crystallographic analysis.     

Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

In situ maleimide bridging of disulfides and a new approach to protein PEGylation

The introduction of non-natural entities into proteins by chemical modification has numerous applications in fundamental biological science and for the development and manipulation of peptide and protein therapeutics. The reduction of native disulfide bonds provides a convenient method to access two nucleophilic cysteine residues that can serve as ideal attachment points for such chemical modification. The optimum bioconjugation strategy utilizing these cysteine residues should include the reconstruction of a bridge to mimic the role of the disulfide bond, maintaining structure and stability of the protein. Furthermore, the bridging chemical modification should be as rapid as possible to prevent problems associated with protein unfolding, aggregation, or disulfide scrambling. This study reports on an in situ disulfide reduction-bridging strategy that ensures rapid sequestration of the free cysteine residues in a bridge, using dithiomaleimides. This approach is then used to PEGylate the peptide hormone somatostatin and retention of biological activity is demonstrated.     

Identification of a Disulfide Bridge Important for Transport Function of SNAT4 Neutral Amino Acid Transporter

SNAT4 is a member of system N/A amino acid transport family that primarily expresses in liver and muscles and mediates the transport of L-alanine. However, little is known about the structure and function of the SNAT family of transporters. In this study, we showed a dose-dependent inhibition in transporter activity of SNAT4 with the treatment of reducing agents, dithiothreitol (DTT) and Tris(2-carboxyethyl)phosphine (TCEP), indicating the possible involvement of disulfide bridge(s). Mutation of residue Cys-232, and the two highly conserved residues Cys-249 and Cys-321, compromised the transport function of SNAT4. However, this reduction was not caused by the decrease of SNAT4 on the cell surface since the cysteine-null mutant generated by replacing all five cysteines with alanine was equally capable of being expressed on the cell surface as wild-type SNAT4. Interestingly, by retaining two cysteine residues, 249 and 321, a significant level of L-alanine uptake was restored, indicating the possible formation of disulfide bond between these two conserved residues. Biotinylation crosslinking of free thiol groups with MTSEA-biotin provided direct evidence for the existence of a disulfide bridge between Cys-249 and Cys-321. Moreover, in the presence of DTT or TCEP, transport activity of the mutant retaining Cys-249 and Cys-321 was reduced in a dose-dependent manner and this reduction is gradually recovered with increased concentration of H₂O₂. Disruption of the disulfide bridge also decreased the transport of L-arginine, but to a lesser degree than that of L-alanine. Together, these results suggest that cysteine residues 249 and 321 form a disulfide bridge, which plays an important role in substrate transport but has no effect on trafficking of SNAT4 to the cell surface.     

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.     

The crystal structure of TrxA(CACA): Insights into the formation of a [2Fe-2S] iron-sulfur cluster in an Escherichia coli thioredoxin mutant

Escherichia coli thioredoxin is a small monomeric protein that reduces disulfide bonds in cytoplasmic proteins. Two cysteine residues present in a conserved CGPC motif are essential for this activity. Recently, we identified mutations of this motif that changed thioredoxin into a homodimer bridged by a [2Fe-2S] iron-sulfur cluster. When exported to the periplasm, these thioredoxin mutants could restore disulfide bond formation in strains lacking the entire periplasmic oxidative pathway. Essential for the assembly of the iron-sulfur was an additional cysteine that replaced the proline at position three of the CGPC motif. We solved the crystalline structure at 2.3 Angstroms for one of these variants, TrxA(CACA). The mutant protein crystallized as a dimer in which the iron-sulfur cluster is replaced by two intermolecular disulfide bonds. The catalytic site, which forms the dimer interface, crystallized in two different conformations. In one of them, the replacement of the CGPC motif by CACA has a dramatic effect on the structure and causes the unraveling of an extended alpha-helix. In both conformations, the second cysteine residue of the CACA motif is surface-exposed, which contrasts with wildtype thioredoxin where the second cysteine of the CXXC motif is buried. This exposure of a pair of vicinal cysteine residues apparently allows thioredoxin to acquire an iron-sulfur cofactor at its active site, and thus a new activity and mechanism of action.     

Deswapping bovine odorant binding protein

The X-ray structure of bovine Odorant Binding Protein (bOBP) revealed its association as a domain swapped dimer. bOBP, devoid of any cysteines, contrasts with other mammalian OBPs, which are monomeric and possess at least one disulfide bridge. We have produced a mutant of bOBP in which a glycine residue was inserted after position 121. This mutation yielded a monomeric bOBP-121Gly+ in which domain swapping has been reverted. Here, we have subsequently introduced two mutations, Trp64Cys and His155Cys, in view to stabilize the putative monomer with a disulfide bridge. We have determined the crystal structure of this triple mutant at 1.65 A resolution. The mutant protein is monomeric, stabilized by a disulfide bridge between Trp64Cys and His155Cys, with a backbone superimposable to that of native bOBP, with the exception of the hinge and of the 10 residues at the C-terminus. bOBP triple mutant binds 1-amino-anthracene, 1-octen-3-ol (bOBP co-purified ligand) and other ligands with microM Kd values comparable to those of the swapped dimer.     

Redox regulation of Cdc25C

The Cdc25 family of dual specific phosphatases are critical components of cell cycle progression and checkpoint control. Certain stresses such as ultraviolet light stimulate the rapid and selective destruction of Cdc25A protein through a Chk1 protein kinase-dependent pathway. We demonstrate that in contrast to cellular stresses previously examined, hydrogen peroxide exposure affects Cdc25C but not Cdc25A levels. Pharmacological inhibition of Chk1 activity or a mutant of Cdc25C that lacks the Chk1 phosphorylation site still undergoes degradation in response to oxidants. We also demonstrate that in vitro hydrogen peroxide stimulates an intramolecular disulfide bond between the active site cysteine at position 377 and another invariant cysteine at position 330. The in vivo stability of Cdc25C is substantially reduced by the mutation of either of these two cysteine residues. In contrast, a double (C2) mutant of both cysteine 330 and cysteine 377 results in a protein that is more stable than wild type Cdc25C and is resistant to oxidative stress-induced degradation. In addition, the C2 mutant, which is unable to form an intramolecular disulfide bond, has reduced binding to 14-3-3 in vitro and in vivo. These results suggest that oxidative stress may induce cell cycle arrest in part through the degradation of Cdc25C.     

Site-specific mutagenesis of the human interleukin-1 beta gene: structure-function analysis of the cysteine residues

Human interleukin-1 beta (IL-1 beta) has two cysteines located at amino acid residues 8 and 71 of the mature protein consisting 153 amino acids. To clarify the role of these characteristic cysteine residues in IL-1 beta, at first, an expression plasmid for site-specific mutagenesis has been constructed by inserting the ori and intergenic region of phage f1 into the IL-1 beta expression vector. The plasmid can be used not only for isolation of the modified IL-1 beta gene but for expression of the mutant protein in Escherichia coli. Using this plasmid, each of the cysteine codons in IL-1 beta gene was changed to serine or alanine codon, or deleted. The modified IL-1 beta showed that the two cysteine residues in IL-1 beta are not essential for biological activity but not to be eliminated for the maintenance of the functional structure of IL-1 beta.     

Structure of oxidized bacteriophage T4 glutaredoxin (thioredoxin). Refinement of native and mutant proteins.

The structure of wild-type bacteriophage T4 glutaredoxin (earlier called thioredoxin) in its oxidized form has been refined in a monoclinic crystal form at 2.0 A resolution to a crystallographic R-factor of 0.209. A mutant T4 glutaredoxin gives orthorhombic crystals of better quality. The structure of this mutant has been solved by molecular replacement methods and refined at 1.45 A to an R-value of 0.175. In this mutant glutaredoxin, the active site residues Val15 and Tyr16 have been substituted by Gly and Pro, respectively, to mimic that of Escherichia coli thioredoxin. The main-chain conformation of the wild-type protein is similar in the two independently determined molecules in the asymmetric unit of the monoclinic crystals. On the other hand, side-chain conformations differ considerably between the two molecules due to heterologous packing interactions in the crystals. The structure of the mutant protein is very similar to the wild-type protein, except at mutated positions and at parts involved in crystal contacts. The active site disulfide bridge between Cys14 and Cys17 is located at the first turn of helix alpha 1. The torsion angles of these residues are similar to those of Escherichia coli thioredoxin. The torsion angle around the S-S bond is smaller than that normally observed for disulfides: 58 degrees, 67 degrees and 67 degrees for wild-type glutaredoxin molecule A and B and mutant glutaredoxin, respectively. Each sulfur atom of the disulfide cysteines in T4 glutaredoxin forms a hydrogen bond to one main-chain nitrogen atom. The active site is shielded from solvent on one side by the beta-carbon atoms of the cysteine residues plus side-chains of residues 7, 9, 21 and 33. From the opposite side, there is a cleft where the sulfur atom of Cys14 is accessible and can be attacked by a nucleophilic thiolate ion in the initial step of the reduction reaction.     

Structure-function analysis of human interleukin-2. Identification of amino acid residues required for biological activity

To locate functional domains of the interleukin-2 (IL-2) protein, a cDNA clone encoding biologically active human IL-2 was mutagenized using synthetic oligonucleotides to incorporate defined amino acid substitutions and deletions in the mature protein. The IL-2 analogs were then produced in Escherichia coli and assayed for the ability to induce proliferation of IL-2-dependent cells and the ability to compete for binding to the IL-2 receptor. Our analysis of over 50 different mutations demonstrated that the integrity of at least three regions of the IL-2 molecule is required for full biological activity: the NH2 terminus (residues 1-20), the COOH terminus (residues 121-133), and 2 of the 3 cysteine residues (58 and 105). Deletion of the NH2-terminal 20 amino acids or the COOH-terminal 10 amino acids resulted in the loss of greater than 99% of bioactivity and binding. Amino acid substitutions at specific positions in these regions also resulted in proteins which retained less than 1% activity. The NH2 terminus and an adjacent internal region were recognized by neutralizing anti-IL-2 antibodies. In combination with the results from epitope competition analysis with neutralizing antibodies, these data are consistent with the IL-2 protein being folded such that the NH2 terminus, the COOH terminus, and the internal 30- to 60-region are juxtaposed to form the binding site recognized by the IL-2 receptor.     

Formation of engineered intersubunit disulfide bond in cytochrome bc1 complex disrupts electron transfer activity in the complex

Protein domain movement of the Rieske iron-sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c1 [S141C(ISP)/G180C(cyt c1)]. The formation of a disulfide bond between ISP and cyt c1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with beta-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc1 complex. Formation of the disulfide bond between ISP and cyt c1 shortens the distance between the [2Fe-2S] cluster and heme c1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.     

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).     

Stabilization by a disulfide bond of the N-terminal domain of a mutant troponin C (TnC48/82)

The regulatory activity of troponin C is reversibly inhibited by a disulfide bridge between cysteine residues introduced by site-directed mutagenesis in positions 48 and 82 (TnC48/82) in the N-terminal domain of rabbit skeletal troponin C (sTnC; Grabarek, Z., Tan, R.-Y., Tao, T., and Gergely, J. (1990) Nature 345, 132-135). In the present work we have investigated the effects of the disulfide on structural properties of TnC48/82 monitored by CD spectroscopy and limited trypsinolysis. The CD spectra of the mutant protein in the oxidized form (oxTnC48/82) with and without Ca2+ are similar to the corresponding ones of the reduced and carboxamidomethylated form (CAMTnC48/82), indicating that the disulfide has essentially no effect on the overall secondary structure. The N-terminal domain of oxTnC48/82 is resistant to thermal unfolding, but that of CAMTnC48/82 is only slightly more stable than the corresponding domain of sTnC. In the presence of Ca2+ oxTnC48/82 is more resistant to trypsinolysis than sTnC whereas the rate of tryptic digestion of CAMTnC48/82 is the same as that of sTnC, indicating that peptide bonds adjacent to lysine residues at position 84 and 88, the sites of tryptic attack, are protected by the disulfide. The disulfide cross-linked N-terminal peptide of TnC48/82 does not bind TnI, unlike its reduced or carboxamidomethylated forms. Our data indicate that the disulfide between Cys48 and Cys82 stabilizes the structure of the N-terminal domain of TnC and blocks its ability to interact with TnI. The effects of the disulfide appear to be restricted to the N-terminal domain of TnC.