Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).     

Two new structured intermediates in the oxidative folding of RNase A

Two new three-disulfide intermediates have been found to be populated in the oxidative folding pathway of bovine pancreatic ribonuclease A at a low temperature (15 degrees C). These intermediates, des-[26-84] and des-[58-110], possess all but one of the four native disulfide bonds and have a stable tertiary structure, similar to the two previously observed intermediates, des-[65-72] and des-[40-95]. While the latter two des species each lack one surface-exposed disulfide bond, the newly discovered intermediates each lack one buried disulfide bond. The possible involvement of these species in the rate-determining steps during the oxidative folding of RNase A is discussed and a specific role for such species during oxidative folding is suggested.     

Catalysis of the oxidative folding of bovine pancreatic ribonuclease A by protein disulfide isomerase

The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 degrees C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 microM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 degrees C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.     

Slow folding of three-fingered toxins is associated with the accumulation of native disulfide-bonded intermediates.

Snake neurotoxins are short all-beta proteins that display a complex organization of the disulfide bonds: two bonds connect consecutive cysteine residues (C43-C54, C55-C60), and two bonds intersect when bridging (C3-C24, C17-C41) to form a particular structure classified as "disulfide beta-cross". We investigated the oxidative folding of a neurotoxin variant, named alpha62, to define the chemical nature of the three-disulfide intermediates that accumulate during the process in order to describe in detail its folding pathway. These folding intermediates were separated by reverse-phase HPLC, and their disulfide bonds were identified using a combination of tryptic hydrolysis, manual Edman degradation, and mass spectrometry. Two dominant intermediates containing three native disulfide bonds were identified, lacking the C43-C54 and C17-C41 pairing and therefore named des-[43-54] and des-[17-41], respectively. Both species were individually allowed to reoxidize under folding conditions, showing that des-[17-41] was a fast-forming nonproductive intermediate that had to interconvert into the des-[43-54] isomer before forming the native protein. Conversely, the des-[43-54] intermediate appeared to be the immediate precursor of the oxidized neurotoxin. A kinetic model for the folding of neurotoxin alpha62 which fits with the observed time-course accumulation of des-[17-41] and des-[43-54] is proposed. The effect of turn 2, located between residues 17 and 24, on the overall kinetics is discussed in view of this model.     

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.     

Crystal structures of two mutants that have implications for the folding of bovine pancreatic ribonuclease A.

The Tyr92-Pro93 peptide group of bovine pancreatic ribonuclease A (RNase A) exists in the cis conformation in the native state. From unfolding/refolding kinetic studies of the disulfide-intact wild-type protein and of a variant in which Pro93 had been replaced by Ala, it had been suggested that the Tyr92-Ala93 peptide group also exists in the cis conformation in the native state. Here, we report the crystal structure of the P93A variant. Although there is disorder in the region of residues 92 and 93, the best structural model contains a cis peptide at this position, lending support to the results of the kinetics experiments. We also report the crystal structure of the C[40, 95]A variant, which is an analog of the major rate-determining three-disulfide intermediate in the oxidative folding of RNase A, missing the 40-95 disulfide bond. As had been detected by NMR spectroscopy, the crystal structure of this analog shows disorder in the region surrounding the missing disulfide. However, the global chain fold of the remainder of the protein, including the disulfide bond between Cys65 and Cys72, appears to be unaffected by the mutation.     

Conformational unfolding studies of three-disulfide mutants of bovine pancreatic ribonuclease A and the coupling of proline isomerization to disulfide redox reactions

The equilibrium stability and conformational unfolding kinetics of the [C40A, C95A] and [C65S, C72S] mutants of bovine pancreatic ribonuclease A (RNase A) have been studied. These mutants are analogues of two nativelike intermediates, des[40-95] and des[65-72], whose formation is rate-limiting for oxidative folding and reductive unfolding at 25 degrees C and pH 8.0. Upon addition of guanidine hydrochloride, both mutants exhibit a fast conformational unfolding phase when monitored by absorbance and fluorescence, as well as a slow phase detected only by fluorescence which corresponds to the isomerizations of Pro93 and Pro114. The amplitudes of the slow phase indicate that the two prolines, Pro93 and Pro114, are fully cis in the folded state of the mutants and furthermore that the 40-95 disulfide bond is not responsible for the quenching of Tyr92 fluorescence observed in the slow unfolding phase, contrary to an earlier proposal [Rehage, A., and Schmid, F. X. (1982) Biochemistry 21, 1499-1505]. The ratio of the kinetic unfolding m value to the equilibrium m value indicates that the transition state for conformational unfolding in the mutants exposes little solvent-accessible area, as in the wild-type protein, indicating that the unfolding pathway is not dramatically altered by the reduction of the 40-95 or 65-72 disulfide bond. The stabilities of the folded mutants are compared to that of wild-type RNase A. These stabilities indicate that the reduction of des[40-95] to the 2S species is rate-limited by global conformational unfolding, whereas that of des[65-72] is rate-limited by local conformational unfolding. The isomerization of Pro93 may be rate-limiting for the reduction of the 40-95 disulfide bond in the native protein and in the des[65-72] intermediate.     

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.     

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.     

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.     

Isomers of epidermal growth factor with Ser --> Cys mutation at the N-terminal sequence: isomerization, stability, unfolding, refolding, and structure

The structure of human epidermal growth factor (EGF, 53 amino acids) comprises three distinct loops (A, B, and C) connected correspondingly by the three native disulfide bonds, Cys(6)-Cys(20), Cys(14)-Cys(31), and Cys(33)-Cys(42). The connection of Cys(6) and Cys(20) forming the N-terminal A loop is essential for the biological activity of EGF [Barnham et al. (1998) Protein Sci. 7, 1738-1749] and has also been shown to represent a major kinetic trap in the oxidative folding of EGF [Chang et al. (2001) J. Biol. Chem. 276, 4845-4852]. To further understand the chemical nature of this kinetic trap, we have prepared three EGF mutants each with a single Ser --> Cys mutation at Ser residues (Ser(2), Ser(4), and Ser(9)) flanking Cys(6). This allows competition between Cys(6) and mutated Cys(2), Cys(4), and Cys(9) to link with Cys(20) and to form EGF isomers containing different sizes of the A loop. The results show that, in the cases of EGF(S2C) and EGF(S4C), native Cys(6)-Cys(20) is favored over Cys(2)-Cys(20) and Cys(4)-Cys(20) by 4.5- and 9-fold, respectively, in the state of equilibrium. However, in the case of EGF(S9C), a non-native Cys(9)-Cys(20) is thermodynamically more stable than the native Cys(6)-Cys(20) by a free-energy difference (DeltaG degrees ) of 1.12 kcal/mol. Implications of these data in the formation of kinetic trap of EGF folding are discussed. Stabilized isomers of EGF were further generated from denaturation of wild-type and mutant EGF via the method of disulfide scrambling. Properties of these diverse isomers of EGF, including their isomerization, stability, unfolding, refolding, and disulfide structures, are described in this paper.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Oxidative folding of bovine pancreatic ribonuclease A: insight into the overall catalysis of the refolding pathway by phosphate

The effects of the strong stabilizing anion, phosphate, on the oxidative folding of bovine pancreatic ribonuclease A were examined. Phosphate was found to catalyze several steps involved in the oxidative folding process at pH 8.0 and 25°C, resulting in an increase in the rate of pre-equilibration of unstructured species on the folding pathway. In the presence of 400 mM phosphate, the overall increase in the rate of regeneration of native protein was caused primarily by the increased formation and stabilization of tertiary structure in the nativelike intermediates, des-[40-95] and des-[65-72], involved in the rate-determining step. Based on the regeneration of native protein and the stability of Cys Ala substituted mutant analogs of the des-species, (C40A, C95A) and (C65A, C72A), it is suggested that the primary role of phosphate is to catalyze the overall regeneration of native protein through nonspecific electrostatic and hydrogen-bonding effects on the protein and solvent.     

Early events in the disulfide-coupled folding of BPTI.

Recent studies of the refolding of reduced bovine pancreatic trypsin inhibitor (BPTI) have shown that a previously unidentified intermediate with a single disulfide is formed much more rapidly than any other one-disulfide species. This intermediate contains a disulfide that is present in the native protein (between Cys14 and 38), but it is thermodynamically less stable than the other two intermediates with single native disulfides. To characterize the role of the [14-38] intermediate and the factors that favor its formation, detailed kinetic and mutational analyses of the early disulfide-formation steps were carried out. The results of these studies indicate that the formation of [14-38] from the fully reduced protein is favored by both local electrostatic effects, which enhance the reactivities of the Cys14 and 38 thiols, and conformational tendencies that are diminished by the addition of urea and are enhanced at lower temperatures. At 25 degrees C and pH 7.3, approximately 35% of the reduced molecules were found to initially form the 14-38 disulfide, but the majority of these molecules then undergo intramolecular rearrangements to generate non-native disulfides, and subsequently the more stable intermediates with native disulfides. Amino acid replacements, other than those involving Cys residues, were generally found to have only small effects on either the rate of forming [14-38] or its thermodynamic stability, even though many of the same substitutions greatly destabilized the native protein and other disulfide-bonded intermediates. In addition, those replacements that did decrease the steady-state concentration of [14-38] did not adversely affect further folding and disulfide formation. These results suggest that the weak and transient interactions that are often detected in unfolded proteins and early folding intermediates may, in some cases, not persist or promote subsequent folding steps.     

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.     

Identification of formation of initial native structure in onconase from an unfolded state

In the oxidative folding of onconase, the stabilization of intermediates early in the folding process gives rise to efficient formation of its biologically active form. To identify the residues responsible for the initial formation of structured intermediates, the transition from an ensemble of unstructured three-disulfide species, 3S(U), to a single structured three-disulfide intermediate species, des-[30-75] or 3S(F), at pH 8.0 and 25 °C was examined. This transition was first monitored by far-UV circular dichroism spectroscopy at pH 8.0 and 25 °C, showing that it occurs with the formation of secondary structure, presumably because of native interactions. The time dependence of formation of nativelike structure was then followed by nuclear magnetic resonance spectroscopy after we had arrested the transition at different times by lowering the pH to 3 and then acquiring (1)H-(15)N heteronuclear single-quantum coherence spectra at pH 3 and 16 °C to identify amide hydrogens that become part of nativelike structure. H/D exchange was utilized to reduce the intensity of resonances from backbone amide hydrogens not involved in structure, without allowing exchange of backbone amide hydrogens involved in initial structure. Six hydrogen-bonding residues, namely, Tyr38, Lys49, Ser82, Cys90, Glu91, and Ala94, were identified as being involved in the earliest detectable nativelike structure before complete formation of des-[30-75] and are further stabilized later in the formation of this intermediate through S-S/SH interchange. By observing the stabilization of the structures of these residues by their neighboring residues, we have identified the initial, nativelike structural elements formed in this transition, providing details of the initial events in the oxidative folding of onconase.     

Characterization of the fast-forming intermediate, des [30-75], in the reductive unfolding of onconase

A fast-forming intermediate in the reductive unfolding of frog onconase (ONC), des [30-75], analogous to the des [40-95] intermediate found in the reductive unfolding of its structural homologue, bovine pancreatic ribonuclease A (RNase A), has been isolated and characterized. The midpoints of the thermal transition and chemical denaturing curves (representing global unfolding) indicate that the conformation of des [30-75] is considerably less stable than that of the parent molecule, suggesting that the (30-75) disulfide bond plays a significant role in the conformational stability of ONC. While des [30-75] is formed very quickly by a partial reduction of the parent molecule in a local unfolding step, it is not as easily susceptible to further reduction, indicating that its three disulfides are much more buried compared to the (30-75) disulfide bond in the parent protein. The nature of des [30-75] is similar to that of des [40-95] RNase A, in that des [30-75] ONC is also a disulfide-secure species. In addition, based on the resistance to mild reducing conditions, structured des species appear to form in ONC from unstructured three-disulfide-containing ensembles. This step is key in the oxidative folding of RNaseA, and is much faster in ONC than the formation of the structured des [40-95] species in RNase A.     

Characterizing the unstructured intermediates in oxidative folding

A recently developed method is used here to characterize some of the folding intermediates, and the oxidative folding processes, of RNase A. This method is based on the ability of trans-[Pt(en)(2)Cl(2)](2+) to oxidize cysteine residues to form disulfide bonds faster than the disulfide bonds can be rearranged by reshuffling or reduction. Variations of this method have enabled us to address three issues. (i) How the nature of the residual structure and/or conformational order that is present, or develops, during the initial stages of folding can be elucidated. It is shown here that there is a 10-fold increase in the propensity of the unfolded reduced forms of RNase A to form the native set of disulfides directly, compared to the propensity under strongly denaturing conditions (4-6 M GdnHCl). Thus, the unfolded reduced forms of RNase A are not statistical coils with a more condensed form than in the GdnHCl-denatured state; rather, it is suggested that reduced RNase A has a little bias toward a native topology. (ii) The structural characterization of oxidative folding intermediates in terms of disulfide pairing is demonstrated; specifically, a lower-limit estimate is made of the percentage of native disulfide-containing molecules in the two-disulfide ensemble of RNase A. (iii) The critical role of structured intermediate species in determining the oxidative folding pathways of proteins was shown previously. Here, we demonstrate that the presence of a structured intermediate in the oxidative folding of proteins can be revealed by this method.     

Conformational propensities of protein folding intermediates: distribution of species in the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of bovine pancreatic ribonuclease A

A key problem in experimental protein folding is that of characterizing the conformational ensemble of denatured proteins under folding conditions. We address this problem by studying the conformational propensities of reductively unfolded RNase A under folding conditions, since earlier work has indicated that the equilibrium conformational ensemble of fully reduced RNase A resembles the transient conformational ensemble of a burst-phase folding intermediate of disulfide-intact RNase A. To assess these propensities, the relative disulfide-bond populations of the 1S, 2S, and 3S ensembles of the [C40A,C95A] mutant of RNase A were measured. Thirteen of the fifteen possible disulfide bonds are observed, consistent with earlier results and with the rapid reshuffling and lack of stable tertiary structure in these ensembles. This broad distribution contradicts recent observations by another group, but rigorous cross-checks show unambiguously that our data are self-consistent whereas their data are not. The distributions of disulfide bonds in the wild-type and mutant proteins show a power-law dependence on loop length, with an exponent that is significantly smaller than the exponents of either ideal or excluded-volume polymers. The 65-72 disulfide bond is much more strongly favored than would be predicted by this power law, consistent with earlier peptide studies and the disulfide-bond distributions of the 1S and 2S ensembles in wild-type RNase A. Experimental evidence suggests that this preference results from conformational biases in the backbone, rather than from differing accessibilities or reactivities of the two cysteine residues. In general, the other disulfide species do not deviate significantly from the power-law dependence, indicating that the conformational biases are relatively weak.     

Study of a major intermediate in the oxidative folding of leech carboxypeptidase inhibitor: contribution of the fourth disulfide bond

The oxidative folding pathway of leech carboxypeptidase inhibitor (LCI; four disulfide bonds) proceeds through the formation of two major intermediates (III-A and III-B) that contain three native disulfide bonds and act as strong kinetic traps in the folding process. The III-B intermediate lacks the Cys19-Cys43 disulfide bond that links the beta-sheet core with the alpha-helix in wild-type LCI. Here, an analog of this intermediate was constructed by replacing Cys19 and Cys43 with alanine residues. Its oxidative folding follows a rapid sequential flow through one, two, and three disulfide species to reach the native form; the low accumulation of two disulfide intermediates and three disulfide (scrambled) isomers accounts for a highly efficient reaction. The three-dimensional structure of this analog, alone and in complex with carboxypeptidase A (CPA), was determined by X-ray crystallography at 2.2A resolution. Its overall structure is very similar to that of wild-type LCI, although the residues in the region adjacent to the mutation sites show an increased flexibility, which is strongly reduced upon binding to CPA. The structure of the complex also demonstrates that the analog and the wild-type LCI bind to the enzyme in the same manner, as expected by their inhibitory capabilities, which were similar for all enzymes tested. Equilibrium unfolding experiments showed that this mutant is destabilized by approximately 1.5 kcal mol(-1) (40%) relative to the wild-type protein. Together, the data indicate that the fourth disulfide bond provides LCI with both high stability and structural specificity.