Three-disulfide Species Research Articles

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.

Oxidative Folding and Structural Analyses of a Kunitz-Related Inhibitor and Its Disulfide Intermediates: Functional Implications

Tick-derived protease inhibitor (TdPI) is a tight-binding Kunitz-related inhibitor of human tryptase β with a unique structure and disulfide-bond pattern. Here we analyzed its oxidative folding and reductive unfolding by chromatographic and disulfide analyses of acid-trapped intermediates. TdPI folds through a stepwise generation of heterogeneous populations of one-disulfide, two-disulfide, and three-disulfide intermediates, with a major accumulation of the nonnative three-disulfide species IIIa. The rate-limiting step of the process is disulfide reshuffling within the three-disulfide population towards a productive intermediate that oxidizes directly into the native four-disulfide protein. TdPI unfolds through a major accumulation of the native three-disulfide species IIIb and the subsequent formation of two-disulfide and one-disulfide intermediates. NMR characterization of the acid-trapped and further isolated IIIa intermediate revealed a highly disordered conformation that is maintained by the presence of the disulfide bonds. Conversely, the NMR structure of IIIb showed a native-like conformation, with three native disulfide bonds and increased flexibility only around the two free cysteines, thus providing a molecular basis for its role as a productive intermediate. Comparison of TdPI with a shortened variant lacking the flexible prehead and posthead segments revealed that these regions do not contribute to the protein conformational stability or the inhibition of trypsin but are important for both the initial steps of the folding reaction and the inhibition of tryptase β. Taken together, the results provide insights into the mechanism of oxidative folding of Kunitz inhibitors and pave the way for the design of TdPI variants with improved properties for biomedical applications.

Folding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approach

In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo.

Major kinetic traps for the oxidative folding of leech carboxypeptidase inhibitor.

The leech carboxypeptidase inhibitor (LCI) is a 66-amino acid protein, containing four disulfides that stabilize its structure. This polypeptide represents an excellent model for the study and understanding of the diversity of folding pathways in small, cysteine-rich proteins. The pathway of oxidative folding of LCI has been elucidated in this work, using structural and kinetic analysis of the folding intermediates trapped by acid quenching. Reduced and denatured LCI refolds through a rapid, sequential flow of one- and two-disulfide intermediates and reaches a rate-limiting step in which a mixture of three major three-disulfide species and a heterogeneous population of non-native four-disulfide (scrambled) isomers coexist. The three three-disulfide intermediates have been identified as major kinetic traps along the folding pathway of LCI, and their disulfide structures have been elucidated in this work. Two of them contain only native disulfide pairings, and one contains one native and two non-native disulfide bonds. The coexistence of three-disulfide kinetic traps adopting native disulfide bonds together with a significant proportion of fully oxidized scrambled isomers shows that the folding pathway of LCI features properties exhibited by both the bovine pancreatic trypsin inhibitor and hirudin, two diverse models with extreme folding characteristics. The results further demonstrate the large diversity of disulfide folding pathways.

Folding of a disulfide-bonded protein species with free thiol(s): competition between conformational folding and disulfide reshuffling in an intermediate of bovine pancreatic ribonuclease A.

The conformational folding of the nativelike intermediate des-[40-95] on the major oxidative folding pathway of bovine pancreatic ribonuclease A (RNase A) has been examined at various pHs and temperatures in the absence of a redox reagent. Des-[40-95] has three of the four disulfide bonds of native RNase A and lacks the bond between Cys40 and Cys95. This three-disulfide species was unfolded at low pH to inhibit any disulfide reshuffling and was refolded at higher pH, allowing both conformational folding and disulfide-reshuffling reactions to take place. As a result of this competition, 15-85% of des-[40-95], depending on the experimental conditions, undergoes intramolecular disulfide-reshuffling reactions. That portion of the des-[40-95] population which has native isomers of essential proline residues appears to fold faster than the disulfide reaction can occur. However, when the folding is retarded, conceivably by the presence of non-native isomers of essential proline residues, des-[40-95] may reshuffle before completing the conformational folding process. These results enable us to distinguish among current models for the critical structure-forming step in oxidative folding and reveal a new model for coupling proline isomerization to disulfide-bond formation. These experiments also demonstrate that the reshuffling-folding competition assay is a useful tool for detecting structured populations in conformational folding intermediates.

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 °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 μM 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 °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.

Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.

The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.

Regeneration of bovine pancreatic ribonuclease A: detailed kinetic analysis of two independent folding pathways.

The regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, proceeds through two separate pathways in which separate nativelike three-disulfide species are populated. The populations of these two three-disulfide species during the regeneration process have been monitored directly through the use of a reduction pulse. A detailed kinetic analysis of the regeneration process using improved experimental procedures and data analysis has been carried out to obtain rate constants for disulfide interconversion among the various disulfide-bonded intermediates. This analysis indicates that these two pathways can account for essentially 100% of the native protein regenerated and that the relative amount of native protein regenerated through these two pathways is insensitive to the redox conditions used. These results indicate that the rate-determining step in both pathways involves formation of the nativelike three-disulfide species, a step in which most of the conformational folding takes place. The experimentally determined rate constants indicate that these two pathways are sufficient to explain the differences in the temperature dependence of the regeneration rate with different redox reagents. In addition, the population of a fully oxidized species that contains three native disulfide bonded pairs and a dithiothreitol bridging cysteines 65 and 72 has been observed.

Regeneration of bovine pancreatic ribonuclease A: identification of two nativelike three-disulfide intermediates involved in separate pathways.

During the regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, the disulfide-containing protein intermediates achieve a steady-state distribution. By manipulating the redox conditions after the attainment of the steady-state condition, it has been possible to kinetically trap and, thereby, isolate and identify the disulfide-bonded species that follow the rate-determining step in the regeneration pathway. Two three-disulfide species have been identified by peptide mapping. Both species contain three native disulfide-bond pairings, one lacks the 65-72 disulfide bond (des-[65-72]), and the other lacks the 40-95 disulfide bond (des-[40-95]). These species are the same as those identified during the reduction of RNase A. By restarting the regeneration process from isolated des-[65-72] and des-[40-95], it is shown that both intermediates lie directly on regeneration pathways.

Relationship between the optimal temperature for oxidative refolding and the thermal stability of refolded state of hen lysozyme three-disulfide derivatives.

The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural characterization of a three-disulfide intermediate of ribonuclease A involved in both the folding and unfolding pathways.

Earlier studies of the unfolding pathway of native bovine pancreatic ribonuclease A (using dithiothreitol as the reducing agent) revealed that the three-disulfide species lacking the disulfide bond between cysteine 65 and cysteine 72 is the most highly populated intermediate [Rothwarf & Scheraga (1991) J. Am. Chem. Soc. 113, 6293-6294]. This unfolding intermediate is referred to as des-[65-72]-RNase A. In order to determine the role of des-[65-72]-RNase A, i.e. of the 65-72 disulfide bond, in the structural folding/unfolding processes of RNase A, the stability and structure of this unfolding intermediate were determined by examining its thermal transition curve and by using two- and three-dimensional homonuclear 1H NMR spectroscopy. The midpoint of the thermal transition of des-[65-72]-RNase A was found to be 17.8 degrees C lower than that of native RNase A. A set of conformations that are consistent with the NMR-derived constraints was obtained by minimizing, first, a variable-target function and, then, the conformational energy. These conformations exhibit a well-defined structure that is very similar to that of native ribonuclease A in regions where the native protein has a regular backbone structure such as a beta-sheet or a helix. Some of the loop regions of the several computed structures exhibit large deviations from each other as well as from native ribonuclease A. However, these results indicate that des-[65-72]-RNase A has a close structural similarity to RNase A in all regions with the only major differences occurring in a loop region comprising residues 60-72. This led to the conclusion that, in reduction pathways that include des-[65-72]-RNase A (at 25 degrees C, pH 8.0), the rate-determining step corresponds to a partial unfolding event in one region of the protein and not to a global conformational unfolding process. The results further suggest that, in the regeneration pathways involving des-[65-72]-RNase A, the loop region from 60 to 72 is the last to fold.

Regeneration of bovine pancreatic ribonuclease A. 2. Kinetics of regeneration

Analysis of the experimental data of the previous paper [Rothwarf, D. M., & Scheraga, H. A. (1993) Biochemistry (first of four papers in this issue)], using the method of Konishi et al. [Konishi, Y., Ooi, T., & Scheraga, H. A. (1981) Biochemistry 20, 3945-3955; Konishi, Y., Ooi, T., & Scheraga H. A. (1982) Biochemistry 21, 4734-4740], and a discussion of the validity of the steady-state kinetic treatment of the data analyzed here are presented. The analysis reveals that RNase A regenerates with oxidized and reduced dithiothreitol (DTTox and DTTred, respectively) through a rearrangement pathway involving one or more three-disulfide species; i.e., multiple pathways could be involved. This pathway is different from that observed when RNase A is regenerated with oxidized and reduced glutathione (GSSG and GSH, respectively). These differences result primarily from the very different characteristics of the oxidation of thiols with DTTox and GSSG, respectively. In addition, the concept of multiple pathways, as applied to the regeneration of RNase A, is developed.

Regeneration of bovine pancreatic ribonuclease A. 4. Temperature dependence of the regeneration rate

The rate of regeneration of bovine pancreatic ribonuclease A with oxidized and reduced dithiothreitol (DTTox and DTTred, respectively) decreases by a factor of 10 when the temperature is increased from 25 to 37 degrees C. The rate of regeneration of RNase A with oxidized and reduced glutathione increases slightly over that same range of temperature. This suggests that the regeneration processes with the two types of redox reagents proceed through different pathways. There is a significant change in the distribution of three-disulfide intermediates populated during regeneration with DTTox/DTTred over the range of temperature 15-37 degrees C that suggests that the three-disulfide species populated at 15 degrees C are directly involved in the major regeneration pathway observed at 25 degrees C.

Circular dichroism spectroscopy of the intermediates that precede the rate-limiting step of the refolding pathway of bovine pancreatic trypsin inhibitor. Relationship of conformation and the refolding pathway.

Circular dichroism spectra of the partially folded trapped intermediates were measured in order to aid in the elucidation of the conformational forces which determine a nonrandom, nonsequential pathway of disulfide bond formation upon refolding of bovine pancreatic trypsin inhibitor. Whatever conformation was responsible for the kinetic rates of the intermediates should be stabilized by the presence of their trapped disulfide bonds. The near-ultraviolet spectra provide considerable information about the environments of the aromatic and disulfide side chains. The predominant single-disulfide intermediate has significant nonrandom conformation not present in the fully reduced protein, with aromatic rings and the disulfide bond in stabilized asymmetric environments. Forming either of the two nonnative, but kinetically important, second disulfides in this intermediate does not produce unequivocably different conformations. Forming a second native, but kinetically unproductive, disulfide produces a substantial decrease in randomness, which may hinder formation of the third disulfide. The largest conformational changes occur upon disulfide rearrangement to the stable, correctly refolded, two- and three-disulfide species. Interpretation of the far-ultraviolet spectra in terms of the secondary structure of the intermediates is uncertain, due to the atypical spectra of the folded forms of the protein. Consequently, we are unable to determine unambiguously the secondary structure of the intermediates. However, all the spectra show that nonrandom conformations of the polypeptide chain gradually appear as disulfide bond formation progresses, as expected from the nonrandom pathway of the latter.