Impact of an easily reducible disulfide bond on the oxidative folding rate of multi-disulfide-containing proteins.

The burial of native disulfide bonds, formed within stable structure in the regeneration of multi-disulfide-containing proteins from their fully reduced states, is a key step in the folding process, as the burial greatly accelerates the oxidative folding rate of the protein by sequestering the native disulfide bonds from thiol-disulfide exchange reactions. Nevertheless, several proteins retain solvent-exposed disulfide bonds in their native structures. Here, we have examined the impact of an easily reducible native disulfide bond on the oxidative folding rate of a protein. Our studies reveal that the susceptibility of the (40-95) disulfide bond of Y92G bovine pancreatic ribonuclease A (RNase A) to reduction results in a reduced rate of oxidative regeneration, compared with wild-type RNase A. In the native state of RNase A, Tyr 92 lies atop its (40-95) disulfide bond, effectively shielding this bond from the reducing agent, thereby promoting protein oxidative regeneration. Our work sheds light on the unique contribution of a local structural element in promoting the oxidative folding of a multi-disulfide-containing protein.     

Catalysis of the oxidative folding of ribonuclease A by protein disulfide isomerase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase

At low concentrations of a glutathione redox buffer, the protein disulfide isomerase (PDI) catalyzed oxidative renaturation of reduced ribonuclease A exhibits a rapid but incomplete activation of ribonuclease, which precedes the steady-state reaction. This behavior can be attributed to a GSSG-dependent partitioning of the substrate, reduced ribonuclease, between two classes of thiol/disulfide redox forms, those that can be converted to active ribonuclease at low concentrations of GSH and those that cannot. With catalytic concentrations of PDI and near stoichiometric concentrations of glutathione disulfide, approximately 4 equiv (2 equiv of ribonuclease disulfide) of GSH are formed very rapidly followed by a slower formation of GSH, which corresponds to an additional 2 disulfide bond equiv. The rapid formation of RNase disulfide bonds and the subsequent rearrangement of incorrect disulfide isomers to active RNase are both catalyzed by PDI. In the absence of GSSG or other oxidants, disulfide bond equivalents of PDI can be used to form disulfide bonds in RNase in a stoichiometric reaction. In the absence of a glutathione redox buffer, the rate of reduced ribonuclease regeneration increases markedly with increasing PDI concentrations below the equivalence point; however, PDI in excess over stoichiometric concentrations inhibits RNase regeneration.     

RNase activity requires formation of disulfide bonds and is regulated by the redox state

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as -ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.     

A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.     

A structural model of pestivirus E(rns) based on disulfide bond connectivity and homology modeling reveals an extremely rare vicinal disulfide

E(rns) is a pestivirus envelope glycoprotein and is the only known viral surface protein with RNase activity. E(rns) is a disulfide-linked homodimer of 100 kDa; it is found on the surface of pestivirus-infected cells and is secreted into the medium. In this study, the disulfide arrangement of the nine cysteines present in the mature dimer was established by analysis of the proteolytically cleaved protein. Fragments were obtained after digestion with multiple proteolytic enzymes and subsequently analyzed by liquid chromatography-electrospray ionization mass spectrometry. The analysis demonstrates which cysteine is involved in dimerization and reveals an extremely rare vicinal disulfide bridge of unknown function. With the assistance of the disulfide arrangement, a three-dimensional model was built by homology modeling based on the alignment with members of the Rh/T2/S RNase family. Compared to these other RNase family members, E(rns) shows an N-terminal truncation, a large insertion of a cystine-rich region, and a C-terminal extension responsible for membrane translocation. The homology to mammalian RNase 6 supports a possible role of E(rns) in B-cell depletion.     

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.     

A thermoresistant mutant of ribonuclease T1 having three disulfide bonds

Molecular-dynamic calculations predict that, if Tyr24 and Asn84 are each replaced by a Cys residue, it should be possible to form a third disulfide bond in ribonuclease T1 (RNase T1) between these residues, with only minimal conformational changes at the catalytic site. The gene encoding such a mutant variant of RNase T1 (Tyr24----Cys24, Asn84----Cys84) was constructed by the cassette mutagenesis method using a chemically synthesized gene. In order to reduce the toxic effect of the mutant enzyme (RNase T1S) on an Escherichia coli host, we arranged for the protein to be secreted into the periplasmic space by using a vector that harbors a gene for an alkaline phosphatase signal peptide under the control of the trp promoter. The nucleolytic activity of RNase T1S toward pGpC was approximately the same as that of RNase T1 at 37 degrees C (pH 7.5). Moreover, at 55 degrees C, RNase T1S retained nearly 70% of its activity while the activity of the wild-type enzyme was reduced to less than 10%. RNase T1S was also more resistant to denaturation by urea than the wild-type enzyme. However, unlike RNase T1, RNase T1S was irreversibly and almost totally inactivated by boiling at 100 degrees C for 15 min.     

Both PDI and PDIp can attack the native disulfide bonds in thermally-unfolded RNase and form stable disulfide-linked complexes

Protein disulfide isomerase (PDI) and its pancreatic homolog (PDIp) are folding catalysts for the formation, reduction, and/or isomerization of disulfide bonds in substrate proteins. However, the question as to whether PDI and PDIp can directly attack the native disulfide bonds in substrate proteins is still not answered, which is the subject of the present study. We found that RNase can be thermally unfolded at 65°C under non-reductive conditions while its native disulfide bonds remain intact, and the unfolded RNase can refold and reactivate during cooling. Co-incubation of RNase with PDI or PDIp during thermal unfolding can inactivate RNase in a PDI/PDIp concentration-dependent manner. The alkylated PDI and PDIp, which are devoid of enzymatic activities, cannot inactivate RNase, suggesting that the inactivation of RNase results from the disruption of its native disulfide bonds catalyzed by the enzymatic activities of PDI/PDIp. In support of this suggestion, we show that both PDI and PDIp form stable disulfide-linked complexes only with thermally-unfolded RNase, and RNase in the complexes can be released and reactivated dependently of the redox conditions used. The N-terminal active site of PDIp is essential for the inactivation of RNase. These data indicate that PDI and PDIp can perform thiol-disulfide exchange reactions with native disulfide bonds in unfolded RNase via formation of stable disulfide-linked complexes, and from these complexes RNase is further released.     

Role of the [65-72] disulfide bond in oxidative folding of bovine pancreatic ribonuclease A

To assess the role of the [65-72] disulfide bond in the oxidative folding of RNase A, use has been made of [C65S, C72S], a three-disulfide-containing mutant of RNase A which regenerates from its two-disulfide precursor in an oxidation and conformational folding-coupled rate-determining step. The distribution of disulfide bonds in the one-disulfide-containing ensemble of this mutant has been characterized. In general, the disulfide-bond distribution in its 1S ensemble agrees relatively well with the corresponding distribution in wt-RNase A and with distributions based on calculations of loop entropy, except for the absence of the [65-72] disulfide bond. There is no bias (over the entropic influence) for the three native disulfide bonds, [26-84], [40-95], and [58-110]. Previous oxidative folding results for wt-RNase A indicated the predominance of the des [40-95] intermediate over des [65-72] after the rate-determining step in the regeneration process. Considering that there is no preferential distribution of disulfides in the 1S ensemble of [C65S, C72S], in contrast to the preferential population of the [65-72] disulfide bond in wt-RNase A, these results indicate a critical role for the [65-72] disulfide bond in the regeneration of wt-RNase A. Furthermore, analysis of the disulfide distribution of the 1S intermediates of [C65S, C72S] compared to that of wt-RNase A lends support for a physicochemical basis for the previously observed slow folding rate of this mutant, compared to its analogue (des [65-72]) of wt-RNase A.     

In vivo substrate specificity of periplasmic disulfide oxidoreductases

In Escherichia coli, a family of periplasmic disulfide oxidoreductases catalyzes correct disulfide bond formation in periplasmic and secreted proteins. Despite the importance of native disulfide bonds in the folding and function of many proteins, a systematic investigation of the in vivo substrates of E. coli periplasmic disulfide oxidoreductases, including the well characterized oxidase DsbA, has not yet been performed. We combined a modified osmotic shock periplasmic extract and two-dimensional gel electrophoresis to identify substrates of the periplasmic oxidoreductases DsbA, DsbC, and DsbG. We found 10 cysteine-containing periplasmic proteins that are substrates of the disulfide oxidase DsbA, including PhoA and FlgI, previously established DsbA substrates. This technique did not detect any in vivo substrates of DsbG, but did identify two substrates of DsbC, RNase I and MepA. We confirmed that RNase I is a substrate of DsbC both in vivo and in vitro. This is the first time that DsbC has been shown to affect the in vivo function of a native E. coli protein, and the results strongly suggest that DsbC acts as a disulfide isomerase in vivo. We also demonstrate that DsbC, but not DsbG, is critical for the in vivo activity of RNase I, indicating that DsbC and DsbG do not function identically in vivo. The absence of substrates for DsbG suggests either that the in vivo substrate specificity of DsbG is more limited than that of DsbC or that DsbG is not active under the growth conditions tested. Our work represents one of the first times the in vivo substrate specificity of a folding catalyst system has been systematically investigated. Because our methodology is based on the simple assumption that the absence of a folding catalyst should cause its substrates to be present at decreased steady-state levels, this technique should be useful in analyzing the substrate specificity of any folding catalyst or chaperone for which mutations are available.     

Selective inhibition of protein disulfide isomerase by estrogens

Protein disulfide isomerase (PDI) is a multifunctional microsomal enzyme that participates in the formation of protein disulfide bonds. PDI catalyzes the reduction of protein disulfide bonds in the presence of excess reduced glutathione and has been implicated in the reductive degradation of insulin; E. coli thioredoxin is homologous to two regions in PDI and can also degrade insulin. PDI activity, measured by 125I-insulin degradation or reactivation of randomly oxidized RNase in the presence of reduced glutathione, is non-competitively inhibited by estrogens; half-maximal inhibition was observed at approximately 100 nM estrogen. Other steroid hormones at 1 microM had little or no effect. PDI segment 120-163 (which corresponds to exon 3 of the PDI gene) and 182-230 have significant similarity with estrogen receptor segments 350-392 and 304-349, respectively, located in the estrogen binding domain but not with the steroid domains of the progesterone and glucocorticoid receptors or with thioredoxin, which is insensitive to estrogens. We propose the hypothesis that enzymes can acquire sensitivity to a hormone via exon shuffling to the enzyme gene from the DNA region coding for the hormone binding domain of the hormone's receptor.     

Conserved residues flanking the thiol/disulfide centers of protein disulfide isomerase are not essential for catalysis of thiol/disulfide exchange.

Protein disulfide isomerase (PDI) catalyzes the oxidative folding of proteins containing disulfide bonds by increasing the rate of disulfide bond rearrangements which normally occur during the folding process. The amino acid sequences of the N- and C-terminal redox active sites (PWCGHCK) in PDI are completely conserved from yeast to man and display considerable identity with the redox-active center of thioredoxin (EWCGPCK). Available data indicate that the two thiol/disulfide centers of PDI can function independently in the isomerase reaction and that the cysteine residues in each active site are essential for catalysis. To evaluate the role of residues flanking the active-site cysteines of PDI in function, a variety of mutations were introduced into the N-terminal active site of PDI within the context of both a functional C-terminal active site and an inactive C-terminal active site in which serine residues replaced C379 and C382. Replacement of non-cysteine residues (W34 to Ser, G36 to Ala, and K39 to Arg) resulted in only a modest reduction in catalytic activity in both the oxidative refolding of RNase A and the reduction of insulin (10-27%), independent of the status of the C-terminal active site. A somewhat larger effect was observed with the H37P mutation where approximately 80% of the activity attributable to the N-terminal domain (approximately 40%) was lost. However, the H37P mutant N-terminal site expressed within the context of an inactive C-terminal domain exhibits 30% activity, approximately 70% of the activity of the N-terminal site alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilization of human RNase 1 by introduction of a disulfide bond between residues 4 and 118

In order to stabilize human RNase 1 by introduction of an intramolecular cross-link, a mutant protein (4-118CL RNase 1), in which Arg4 and Val118 are replaced with cysteine residues and linked by a disulfide bond, was designed and expressed in Escherichia coli as inclusion bodies. The 4-118CL RNase 1 that refolded under redox conditions was a monomer without free SH groups and retained 11% of the activity of the wild-type recombinant RNase 1, indicating that the mutant enzyme was correctly folded with the formation of an additional disulfide bond between Cys4 and Cys118. From guanidium chloride denaturation experiments based on the assumption of a two-state transition for unfolding, it was demonstrated that the introduction of the present cross-link increased the thermodynamic stability of RNase 1 by 2.0 kcal/mol. This value was lower than that, 5.4 kcal/mol, theoretically calculated from the reduction of chain entropy of the unfolded state due to the introduction of the cross-link. These results suggest that the present cross-link also destabilized the folded state of RNase 1 by 3.4 kcal/mol. Along with the increase in the thermodynamic stability, the stability of RNase 1 against trypsin digestion was also significantly increased by the introduction of this cross-link. It is likely, although not proven, that stabilized human RNases are favorable for clinical use, because human RNase-based immunotoxins should have long half-lives as to proteolytic degradation after endocytosis.     

Sulfhydryl oxidase from egg white. A facile catalyst for disulfide bond formation in proteins and peptides.

Both metalloprotein and flavin-linked sulfhydryl oxidases catalyze the oxidation of thiols to disulfides with the reduction of oxygen to hydrogen peroxide. Despite earlier suggestions for a role in protein disulfide bond formation, these enzymes have received comparatively little general attention. Chicken egg white sulfhydryl oxidase utilizes an internal redox-active cystine bridge and a FAD moiety in the oxidation of a range of small molecular weight thiols such as glutathione, cysteine, and dithiothreitol. The oxidase is shown here to exhibit a high catalytic activity toward a range of reduced peptides and proteins including insulin A and B chains, lysozyme, ovalbumin, riboflavin-binding protein, and RNase. Catalytic efficiencies are up to 100-fold higher than for reduced glutathione, with typical K(m) values of about 110-330 microM/protein thiol, compared with 20 mM for glutathione. RNase activity is not significantly recovered when the cysteine residues are rapidly oxidized by sulfhydryl oxidase, but activity is efficiently restored when protein disulfide isomerase is also present. Sulfhydryl oxidase can also oxidize reduced protein disulfide isomerase directly. These data show that sulfhydryl oxidase and protein disulfide isomerase can cooperate in vitro in the generation and rearrangement of native disulfide pairings. A possible role for the oxidase in the protein secretory pathway in vivo is discussed.     

Effect of mutation of proline 93 on redox unfolding/folding of bovine pancreatic ribonuclease A.

Both the reductive unfolding and oxidative regeneration of a P93A mutant and wild-type RNase A have been studied at 15 degrees C and pH 8.0. The rate of reduction of the 40--95 disulfide bond is accelerated about 120-fold by the P93A mutation, while the reduction of the 65--72 disulfide bond is not accelerated by this mutation (within the experimental error). Moreover, the reduction of native P93A to des[40--95] is about 10 times faster than the further reduction of the same des[40--95] species. These results demonstrate that the reduction of the mutant proceeds through a local unfolding event and provides strong support for our model in which the reduction of wild-type RNase A to the des species proceeds through two independent local conformational unfolding events. The oxidative regeneration rate of the P93A mutant is comparable to that of wild-type RNase A, suggesting that a cis 92--93 peptide group that is present in native wild-type RNase A and in native des[40--95], is not obligatory for the formation of the third (final) native disulfide bond of des[40--95] by reshuffling from an unstructured 3S precursor. Thus, the trans to cis isomerization of the Tyr92-Pro93 peptide group during the regeneration of wild-type RNase A may occur after the formation of the third native disulfide bond.     

The oxidase DsbA folds a protein with a nonconsecutive disulfide

One of the last unsolved problems of molecular biology is how the sequential amino acid information leads to a functional protein. Correct disulfide formation within a protein is hereby essential. We present periplasmic ribonuclease I (RNase I) from Escherichia coli as a new endogenous substrate for the study of oxidative protein folding. One of its four disulfides is between nonconsecutive cysteines. In general view, the folding of proteins with nonconsecutive disulfides requires the protein disulfide isomerase DsbC. In contrast, our study with RNase I shows that DsbA is a sufficient catalyst for correct disulfide formation in vivo and in vitro. DsbA is therefore more specific than generally assumed. Further, we show that the redox potential of the periplasm depends on the presence of glutathione and the Dsb proteins to maintain it at-165 mV. We determined the influence of this redox potential on the folding of RNase I. Under the more oxidizing conditions of dsb(-) strains, DsbC becomes necessary to correct non-native disulfides, but it cannot substitute for DsbA. Altogether, DsbA folds a protein with a nonconsecutive disulfide as long as no incorrect disulfides are formed.     

Aromatic thiol pKa effects on the folding rate of a disulfide containing protein

The production of proteins via recombinant DNA technology often requires the in vitro folding of inclusion bodies, which are protein aggregates. To create a more efficient redox buffer for the in vitro folding of disulfide containing proteins, aromatic thiols were investigated for their ability to increase the folding rate of scrambled RNase A. Scrambled RNase A is fully oxidized RNase A with a relatively random distribution of disulfide bonds. The importance of the thiol pK(a) value was investigated by the analysis of five para-substituted aromatic thiols with pK(a) values ranging from 5.2 to 6.6. Folding was measured at pH 6.0 where the pK(a) value of the thiols would be higher, lower, or equal to the solution pH. Thus, relative concentrations of thiol and thiolate would vary across the series. At pH 6.0, the aromatic thiols increased the folding rate of RNase A by a factor of 10-23 over that observed for glutathione, the standard additive. Under optimal conditions, the apparent rate constant increased as the thiol pK(a) value decreased. Optimal conditions occurred when the concentration of protonated thiol in solution was approximately 2 mM, although the total thiol concentration varied considerably. The importance of the concentration of protonated thiol in solution can be understood based on equilibrium effects. Kinetic studies suggest that the redox buffer participates as the nucleophile and/or the center thiol in the key rate determining thiol disulfide interchange reactions that occur during protein folding. Aromatic thiols proved to be kinetically faster and more versatile than classical aliphatic thiol redox buffers.     

Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.     

Contribution of individual disulfide bonds to the oxidative folding of ribonuclease A

The eight cysteine residues of ribonuclease A form four disulfide bonds in the native protein. We have analyzed the folding of three double RNase A mutants (C65A/C72A, C58A/C110A, and C26A/C84A, lacking the C65-C72, C58-C110, and C26-C84 disulfide bonds, respectively) and two single mutants (C110A and C26A), in which a single cysteine is replaced with an alanine and the paired cysteine is present in the reduced form. The folding of these mutants was carried out in the presence of oxidized and reduced glutathione, which constitute the main redox agents present within the ER. The use of mass spectrometry in the analysis of the folding processes allowed us (i) to follow the formation of intermediates and thus the pathway of folding of the RNase A mutants, (ii) to quantitate the intermediates that formed, and (iii) to compare the rates of formation of intermediates. By comparison of the folding kinetics of the mutants with that of wild-type RNase A, the contribution of each disulfide bond to the folding process has been evaluated. In particular, we have found that the folding of the C65A/C72A mutant occurs on the same time scale as that of the wild-type protein, thus suggesting that the removal of the C65-C72 disulfide bond has no effect on the kinetics of RNase A folding. Conversely, the C58A/C110A and C26A/C84A mutants fold much more slowly than the wild-type protein. The removal of the C58-C110 and C26-C84 disulfide bonds has a dramatic effect on the kinetics of RNase A folding. Results described in this paper provide specific information about conformational folding events in the regions involving the mutated cysteine residues, thus contributing to a better understanding of the complex mechanism of oxidative folding.