©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DsbA-mediated Disulfide Bond Formation and Catalyzed Prolyl Isomerization in Oxidative Protein Folding (*)

(Received for publication, November 10, 1994; and in revised form, December 19, 1994)

Christian Frech Franz X. Schmid (§)

From the Biochemisches Laboratorium, Universität Bayreuth, D-95440 Bayreuth, Federal Republic of Germany

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The interrelationship between the acquisition of ordered structure, prolyl isomerization, and the formation of the disulfide bonds in assisted protein folding was investigated by using a variant of ribonuclease T1 (C2S/C10N-RNase T1) with a single disulfide bond and two cis-prolyl bonds as a model protein. The thiol-disulfide oxidoreductase DsbA served as the oxidant for forming the disulfide bond and prolyl isomerase A as the catalyst of prolyl isomerization. Both enzymes are from the periplasm of Escherichia coli.

Reduced C2S/C10N-RNase T1 is unfolded in 0 M NaCl, but native-like folded in geq2 M NaCl. Oxidation of 5 µM C2S/C10N-RNase T1 by 8 µM DsbA (at pH 7.0, 25 °C) is very rapid with a t of about 10 s (the second-order rate constant is 7 times 10^3 sM), irrespective of whether the reduced molecules are unfolded or folded. When they are folded, the product of oxidation is the native protein. When they are denatured, first the disulfide bond is formed in the unfolded protein chains and then the native structure is acquired. This slow reaction is limited in rate by prolyl isomerization and catalyzed by prolyl isomerase. The efficiency of this catalysis is strongly decreased by the presence of the disulfide bond.

Apparently, the rank order of chain folding, prolyl isomerization, and disulfide bond formation can vary in the oxidative folding of ribonuclease T1. Such a degeneracy could generally be an advantage for protein folding both in vitro and in vivo.


INTRODUCTION

The molecular mechanism of protein folding is not well understood at present. Often, extensive secondary structure and some tertiary contacts form very rapidly, followed by a slow step which determines the overall rate of folding to the native state (Roder et al., 1988; Udgaonkar and Baldwin, 1988; Kuwajima, 1989; Kim and Baldwin, 1982, 1990; Baldwin, 1991, 1993; Schmid, 1992; Radford et al., 1992; Ptitsyn, 1992; Fersht, 1993; Evans and Radford, 1994). Folding is, however, not a simple sequential process. Locally formed structure is often unstable or non-native (Dyson et al., 1992; Shin et al., 1993; Waltho et al., 1993; Goldberg and Guillou, 1994) and the high resolving power of the tertiary interactions is necessary to select and stabilize those local structures, which are on the productive pathway to the native state. As a consequence, there should be a close cooperation between the early and the late stages of folding (Go, 1983). For many proteins the slow steps involve the formation of the correct disulfide bonds (Creighton, 1992) and the cis/trans isomerizations of prolyl peptide bonds (Brandts et al., 1975; Schmid, 1992). These rate-limiting steps are assisted in vitro and in vivo by folding enzymes. The thiol-disulfide oxidoreductases (protein disulfide isomerases) accelerate the formation and isomerization of disulfide bonds (Goldberger et al., 1963; Venetianer and Straub, 1963; Freedman, 1992, Freedman et al., 1994), and the prolyl isomerases catalyze the cis/trans isomerizations of prolyl peptide bonds (Fischer et al., 1984; Lang et al., 1987; Schmid, 1993).

It is our aim here to find out how the various processes that occur during assisted protein folding are related with each other. In particular, we ask whether the formation of ordered structure, the introduction of disulfide bonds, and the isomerization of prolyl peptide bonds have to occur in a defined order to warrant productive folding and an efficient function of the folding enzymes.

As a model protein we use the C2S/C10N variant of ribonuclease T1 (C2S/C10N-RNase T1) (^1)from Aspergillus oryzae. Our in vitro experiments are carried out by using the thiol-disulfide oxidoreductase DsbA (Bardwell et al., 1991; Kamitani et al., 1992; Akiyama et al., 1992; Bardwell and Beckwith, 1993; Zapun and Creighton, 1994; Nelson and Creighton, 1994) as the oxidant and prolyl isomerase A (Hayano et al., 1991) as the catalyst of prolyl isomerization. Both are from the periplasm of Escherichia coli.

RNase T1 is a small protein with two disulfide bonds (Cys^2-Cys and Cys^6-Cys) and two cis-prolines (Pro and Pro) (Heinemann and Saenger, 1982; Martinez-Oyanedel et al., 1991; Pace et al., 1991). In the C2S/C10N variant only the Cys^6-Cys disulfide bond is present, which connects the amino and the carboxyl terminus. Wild-type RNase T1 and the C2S/C10N variant fold reversibly (Shirley et al., 1989; Kiefhaber et al., 1990a, 1990b; Mayr et al., 1994). When the disulfide bonds are left intact, intermediates with native-like secondary structure are formed rapidly during refolding, followed by slow steps, which are limited in rate by the trans cis isomerizations of Pro and Pro (Kiefhaber et al., 1990b, 1992a; Mullins et al., 1993).

The reduction of the disulfide bonds of RNase T1 is reversible (Pace and Creighton, 1986; Schönbrunner and Schmid, 1992). Reduced protein molecules are unfolded at pH 7.0, 25 °C in the absence of denaturants, but native-like folded with Pro and Pro in the correct cis conformation when geq2 M NaCl is present (Oobatake et al., 1979; Pace et al., 1988; Mücke and Schmid, 1992, 1994a, 1994b). Thus, the conformation of the reduced protein can be changed by varying the NaCl concentration before the formation of the disulfide bonds is initiated. Cys^6 and Cys close a large loop of 98 residues. The formation of this loop should be coupled tightly with chain folding and strongly depend on changes in the distance between these 2 Cys residues and on their accessibility during folding and oxidation.

We find that the oxidative folding of C2S/C10N-RNase T1 can be extremely rapid when both DsbA and prolyl isomerase are present. An obligatory rank order of structure formation, prolyl isomerization, and disulfide bond formation could not be found.


EXPERIMENTAL PROCEDURES

Materials

The C2S/C10N variant of RNase T1 was purified as described (Mayr and Schmid, 1993; Mayr et al., 1994) from E. coli cells transformed with a plasmid carrying a chemically synthesized gene as described by Quaas et al. (1988). Periplasmic peptidyl-prolyl cis/trans isomerase from E. coli was a gift from N. Takahashi (TONEN Corp. Nishi-Tsurugaoka, Japan). A 1.4 nM solution of this enzyme had an activity of 1 PU/ml when measured as described by Fischer et al.(1984). DsbA from E. coli was a gift from R. Glockshuber (Zürich). GdmCl (ultrapure) and urea (ultrapure) were from Schwarz/Mann, and dithioerythritol (DTE), Hepes (sodium salt) and acetonitrile (HPLC grade) were from Sigma. Dithiothreitol (DTT) was from Fluka (Buchs, Switzerland). All other chemicals were from Merck (Darmstadt, Germany). The concentrations of GdmCl, urea, and NaCl were determined by the refraction of the solutions. The equations correlating the refractive index with the concentrations of GdmCl and urea are given by Pace et al.(1989). The respective equation for the concentrations of NaCl solutions is given by Mücke and Schmid (1994b).

Spectroscopic Methods

For optical measurements a Hitachi F4010 fluorescence spectrometer, a Kontron Uvikon 860 spectrophotometer, and a Jasco J-600A spectropolarimeter were used. The concentrations of the reduced and the oxidized forms of C2S/C10N-RNase T1 were determined spectrophotometrically by using an absorption coefficient of = 21,060 M cm (Takahashi et al., 1970). For DsbA is 21,740 M cm (Zapun et al., 1993).

CD spectra were recorded at a protein concentration of 14 µM (far-UV CD) in 0.1-cm cells and of 18.5 µM (near-UV CD) in 1-cm cells thermostatted at 25 °C. The spectra were measured 10 times and averaged. A scan speed of 20 nm/min, a bandwidth of 1 nm, and a time constant of 1 s were used. All spectra were corrected for the contributions of the respective buffers.

Reduction of C2S/C10N-RNase T1

Typically 2 mg of C2S/C10N-RNase T1 were dissolved in 200 µl of 0.3 M Tris/HCl, pH 8.7, containing 6.0 M GdmCl, 2 mM EDTA, 50 mM DTE and incubated at 25 °C for at least 2 h. The reduction was stopped by adding 15 µl of 3.0 M HCl. The reduced protein was immediately separated from the reagents by gel filtration over a 0.5 times 30-cm Sephadex G-25 column, equilibrated with 10 mM HCl. Fractions containing reduced C2S/C10N-RNase T1 were lyophilized and stored at -20 °C. The content of free thiol groups of the reduced protein was determined by Ellman's assay in the absence of GdmCl, using an absorption coefficient of 14150 M cm (Riddles et al., 1983). 2.1-2.3 free thiol groups per C2S/C10N-RNase T1 molecule were found.

Equilibrium Folding Transition

Reduced C2S/C10N-RNase T1 (0.53 µM) was incubated at 25 °C in the presence of 0.1 M Hepes, 2 mM EDTA, 4 mM DTE, pH 7.0, and varying concentrations of NaCl and of urea for at least 3 h. The fluorescence emission of the samples was measured at 320 nm (10-nm bandwidth) after excitation at 268 nm (1.5-nm bandwidth) in 1 times 1-cm cells. The transition was analyzed by assuming a two-state transition between the folded(N) and the unfolded (U) conformations (Santoro and Bolen, 1988).

Refolding Kinetics

Refolding kinetics were typically initiated by a 100-fold dilution of the reduced protein (in 10 mM HCl) or of the denatured, oxidized protein (in 6.0 M urea, 0.1 M Hepes, 2 mM EDTA, pH 7.0) to the final conditions. The kinetics were followed by the increase in fluorescence at 320 nm (10-nm bandwidth) after excitation at 268 nm (1.5-nm bandwidth) in 1 times 1-cm cells. All kinetic experiments were carried out in 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. Samples with reduced protein contained additionally 4 mM DTE. The final protein concentration was 1 µM. The observed kinetic curves were analyzed as a sum of exponential functions by using the program GraFit 3.0 (Erithacus Software, Staines, United Kingdom).

Oxidation of Reduced C2S/C10N-RNase T1 and Analysis by HPLC

The oxidative folding of C2S/C10N-RNase T1 was initiated by a 50-fold dilution of the reduced and unfolded protein (in 10 mM HCl) to give final concentrations of 5 µM C2S/C10N-RNase T1, 8 µM oxidized DsbA, and 0.4 mM GSSG in 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. In some experiments, additionally 1000 PU/ml prolyl isomerase was present. In the prefolding experiments C2S/C10N-RNase T1 was preincubated in 2.8 M NaCl for at least 1 h to produce the folded reduced protein. Aliquots were removed from the oxidation mixtures after varying time intervals and further disulfide bond formation was stopped by the addition of 0.25 volume of 3.0 M HCl. The reduced and oxidized forms were separated by reversed-phase HPLC on a Kontrosorb 5RP18 25 times 0.46-cm column using a linear gradient from 63 to 59% of solvent A in 33 min at a flow rate of 1 ml/min. Solvent A was 0.1% (v/v) trifluoroacetic acid in water and solvent B was 0.1% (v/v) trifluoroacetic acid in water:acetonitrile (1:9). C2S/C10N-RNase T1 was detected and quantified by the absorbance at 215 nm and at 229 nm.

Reactivation Measurements

After various time intervals aliquots were drawn from the oxidation mixture and diluted 1000-fold with a 40 µM GpC solution in 0.05 M Tris/HCl, 2 mM EDTA, pH 7.5 at 25 °C in the spectrophotometer cell. In addition a 400-fold molar excess of trypsin over C2S/C10N-RNase T1 was present to prevent further folding of unfolded or partially folded molecules during the assay. Native C2S/C10N-RNase T1 is insensitive to trypsin under these conditions (Mayr et al., 1994). The increase in absorbance at 280 nm caused by the cleavage of GpC was recorded for 5 min. The initial slope (DeltaA/min) was used as a measure of the enzymatic activity present after various times of oxidation and refolding. The enzymatic activity of native oxidized protein was measured in control experiments.

Reduction of DsbA by DTT

The reduction of oxidized DsbA by DTT was initiated by rapidly mixing 5.5 µl of 0.2 mM DTT (in 0.1 M sodium acetate, 2 mM EDTA, pH 5.0) with 1495 µl of oxidized DsbA to give final conditions of 0.5 µM DsbA and 0.73 µM DTT in 0.1 M Hepes, 2 mM EDTA, pH 7.0, and 0-3.0 M NaCl at 25 °C. The reduction of DsbA was followed by the increase in fluorescence at 322 nm (10-nm bandwidth) (Wunderlich and Glockshuber, 1993) after excitation at 280 nm (2.5-nm bandwidth). The kinetics of dithiol/disulfide exchange were analyzed as a second-order reaction.


RESULTS

Structure of C2S/C10N-RNase T1 in the Reduced and in the Oxidized State

C2S/C10N-RNase T1 contains only a single disulfide bond, which connects Cys^6 and Cys. The breaking of the other disulfide bond between Cys^2 and Cys by the substitutions C2S and C10N was moderately destabilizing and reduced the Gibbs free energy of denaturation of RNase T1 by about 10 kJ/mol. The three-dimensional structure remained almost unchanged, however. A comparative analysis by two-dimensional NMR of the wild-type protein and the C2S/C10N variant showed that significant differences in the CalphaH and NH resonances are found only for the amino-terminal chain region, where the cysteine residues 2 and 10 are located (Mayr et al., 1994).

The limited solubility of the reduced form of C2S/C10N-RNase T1 precluded an analysis by two-dimensional NMR. Instead, CD spectroscopy in the amide and in the aromatic region was used to characterize the structure of C2S/C10N-RNase T1 in the presence and in the absence of the Cys^6-Cys disulfide bond (Fig. 1). When NaCl is absent, the reduced protein is unfolded in 10 mM Hepes, pH 7.0, as well as in 10 mM HCl, pH 2.0. The protein was kept under these acidic conditions prior to the oxidation experiments, to maintain the thiol groups in the reduced form. In the presence of 2.5 M NaCl the CD spectra of the reduced and the oxidized protein in the amide region are almost identical, suggesting that the two forms of the protein show similar secondary structure.


Figure 1: CD spectra of reduced and of oxidized C2S/C10N-RNase T1 in (A) the far-UV and (B) the near-UV region at 25 °C. All spectra were recorded in 10 mM Hepes, pH 7.0, except spectrum 3, which was measured in 10 mM HCl. The protein concentration was 14 µM in 0.1-cm cells for the far-UV CD and 18.5 µM in 1-cm cells for the near-UV CD. Spectrum 1, folded reduced protein, incubated for 1 h in 2.5 M NaCl; spectrum 2, folded oxidized protein, incubated for 1 h in 2.5 M NaCl; spectrum 3, unfolded reduced protein in 10 mM HCl; spectrum 4, reduced protein in the absence of NaCl at pH 7.0.



In the aromatic region the CD spectra of the two forms are similar in shape as well, but the reduced protein shows a higher ellipticity than the oxidized protein (Fig. 1B) and closely resembles the spectra of the wild-type protein with both disulfide bonds either intact or broken (Mücke and Schmid, 1994b). This agrees with our earlier finding that the selective breaking of the Cys^2-Cys disulfide bond (in the oxidized C2S/C10N variant; Mayr et al., 1994) leads to stronger changes of the CD in the aromatic region than the breaking of both disulfide bonds (in the various reduced or reduced and carboxyymethylated forms). The dihedral angles of the Cys^2-Cys and Cys^6-Cys disulfide bonds in RNase T1 are +78° and -83°, respectively (Martinez-Oyanedel et al., 1991), and therefore their contributions to the CD spectrum of the native wild-type protein probably compensate each other (Neubert and Carmack, 1974). A native-like CD in the aromatic region was also observed for the reduced wild-type protein in the presence of 1.2 M KF (Oobatake et al., 1979). Taken together, the analysis by CD suggests that C2S/C10N-RNase T1 attains a folded conformation in 2.5 M NaCl, which resembles the native state of the oxidized protein in both secondary and tertiary structure.

The folded state of reduced C2S/C10N-RNase T1 is reached in a cooperative transition when the concentration of NaCl is increased. This folding transition, as monitored by the change in the fluorescence of Trp (Fig. 2), shows a midpoint at 0.7 M NaCl. It begins at 0 M NaCl, and therefore, in additional experiments 1 and 2 M urea were added to measure the fluorescence of the unfolded protein. These results indicate that a change of the NaCl concentration provides a powerful means to change the structure and stability of the reduced protein prior to the oxidative formation of the disulfide bond.


Figure 2: Folding transition of reduced C2S/C10N-RNase T1 in 0.1 M Hepes, pH 7.0, 25 °C. The increase in protein fluorescence at 320 nm (excitation at 268 nm) is given as a function of the NaCl concentration. To obtain the base line for the unfolded protein the emissions of samples in the presence of 1.0 and 2.0 M, urea were measured in addition. They are shown on the left side of the figure. The individual samples were incubated for at least 3 h under the final conditions of 0.5 µM C2S/C10N-RNase T1 in 0.1 M Hepes, 2 mM EDTA, 4 mM DTE, and the indicated concentrations of NaCl or of urea. The line represents an analysis of the data according to the two-state model by using the method of Santoro and Bolen(1988). The midpoint of the transition is at 0.7 M NaCl.



Refolding Kinetics of Reduced and of Oxidized C2S/C10N-RNase T1

The kinetics of the NaCl-induced refolding of reduced C2S/C10N-RNase T1 were followed by the increase in fluorescence at 320 nm after a dilution of the protein from 0 M NaCl to 2.5 M NaCl at pH 7.0, 25 °C. Refolding is a slow reaction under these conditions (Fig. 3A), and the entire change in fluorescence occurs apparently in a single first-order reaction with a time constant of 390 s. As in the case of other variants of RNase T1 with broken disulfide bonds (Mücke and Schmid, 1992, 1994a, 1994b), this reaction is limited in rate by the trans cis isomerizations of Pro and Pro. It is very well catalyzed by prolyl isomerase. In the presence of 1000 PU/ml prolyl isomerase we find an 19-fold acceleration and refolding is complete within 100 s (Fig. 3A).


Figure 3: Kinetics of refolding of reduced (A) and oxidized (B) C2S/C10N-RNase T1 in the absence and in the presence of prolyl isomerase at pH 7.0, 25 °C. Refolding was monitored by the increase in fluorescence at 320 nm. The refolding conditions were 1 µM protein in 2.5 M NaCl, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. The refolding solution for the reduced protein contained in addition 4 mM DTE. Traces 1 and 3 were measured in the absence of prolyl isomerase, and traces 2 and 4 were measured in the presence of 1000 PU/ml prolyl isomerase. The refolding of reduced C2S/C10N-RNase T1 in A can be described by a single exponential function yielding time constants of 390 s for the uncatalyzed reaction (trace 1) and of 21 s for the catalyzed reaction (trace 2). The refolding of the oxidized protein in B was biphasic. The slow reaction had a time constant of 550 s and the fast folding reaction a time constant of 65 s (trace 3) and could be accelerated to 290 and 7 s, respectively, by 1000 PU/ml prolyl isomerase (trace 4).



The refolding of the oxidized protein under the same conditions is shown in Fig. 3B. It is a biphasic process with time constants of 550 and 65 s, rather than monophasic as the folding of the reduced form in Fig. 3A. The catalysis of folding by prolyl isomerase is much less efficient when the disulfide bonds are left intact. The addition of 1000 PU/ml prolyl isomerase as in Fig. 3A led to a 9-fold acceleration of the 65-s reaction and to a marginal 2-fold acceleration of the 550-s reaction, which involves the very slow trans cis isomerization of Pro (Fig. 3B). As a consequence, the catalyzed refolding of the oxidized protein still requires about 1000 s to reach completion. Clearly, the presence of the disulfide bond in oxidized C2S/C10N-RNase T1 interferes with the catalysis of folding by prolyl isomerase. This is not a local effect, because the Cys^6-Cys disulfide and the cis-prolines are well separated both in the sequence and in the structure of the folded protein. Rather, as in the folding of the oxidized form of the wild-type protein, partially folded structure forms rapidly when the disulfide bond is present and restricts the access of prolyl isomerase to Pro (Mücke and Schmid, 1994a).

Similar kinetics were observed when the refolding of the oxidized protein was followed either by tryptophan fluorescence or by the regain of the RNase activity (Fig. 4). About 10% of the increase in fluorescence occurred in the dead time of manual mixing (3 s). This fast change originates from the presence of a small amount (2-4%) of fast refolding molecules with correct cis isomers of both Pro and Pro and from the formation of native-like intermediates in the folding of molecules with an incorrect trans isomer only at Pro (Kiefhaber et al., 1992a; Mayr et al., 1994). The following slow increase in fluorescence could be well represented by a sum of two exponential functions with time constants of 28 s (11% amplitude) and 280 s (79% amplitude).


Figure 4: Comparison of the refolding kinetics of oxidized C2S/C10N-RNase T1 as monitored by the increase in fluorescence (circle) and by the regain of enzymatic activity (bullet) at pH 7.0, 25 °C. The protein concentration was 1 µM in 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. The slow refolding as monitored by fluorescence was approximated by the sum of two exponential functions with time constants of 280 s (79% amplitude) and of 28 s (11% amplitude), as shown by the continuous line. Slow reactivation was approximated by a single exponential function with a time constant of 330 s, as shown by the broken line.



The time course of reactivation paralleled the increase in fluorescence (Fig. 4), but it was approximated by a single exponential function with a time constant of 315 ± 30 s, regarding the lower accuracy of the activity measurements. The formation of native C2S/C10N-RNase T1 thus seems to be controlled largely by the very slow reaction, which is correlated with extensive formation of ordered structure and is limited in rate by the trans cis isomerization of Pro (Mayr et al., 1994). The good coincidence of the fluorescence and the activity data in Fig. 4indicates that both probes can be used to follow the formation of the native protein in refolding experiments.

Oxidative Folding of C2S/C10N-RNase T1

Native C2S/C10N-RNase T1 was reduced by DTE under unfolding conditions, desalted, and freeze-dried as described under ``Experimental Procedures.'' The lyophilized reduced protein was dissolved in 10 mM HCl, and oxidative folding was initiated by a 50-fold dilution to a final concentration of 5 µM in the presence of 8 µM oxidized DsbA and 0.4 mM GSSG in 2 mM EDTA, 0.1 M Hepes, pH 7.0 at 25 °C. The time course of the reaction was followed by two probes. To monitor the formation of the Cys^6-Cys disulfide bond during refolding, aliquots were taken, and oxidation was quenched by a rapid acidification to pH 2.0. The reduced and the oxidized forms of C2S/C10N-RNase T1 in these samples were then separated by reversed-phase HPLC (Fig. 5), and their concentrations were determined as a function of the time of refolding.


Figure 5: HPLC elution profile of a mixture of acid-quenched reduced and oxidized C2S/C10N-RNase T1. The mixture in 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C was brought to pH 2 by the addition of 0.25 volumes of 3 M HCl, and about 1 µg of reduced and of oxidized protein was loaded onto the reversed-phase column and eluted as described under ``Experimental Procedures.''



Chain folding and the formation of the native state of the protein was followed by activity assays as in Fig. 4. Further reoxidation during the assay was stopped by the 1000-fold dilution of the protein and the oxidant DsbA, and further reactivation during the assay was inhibited by adding trypsin (Mayr et al., 1994). The increase in RNase T1 fluorescence could not be used to monitor the formation of ordered structure in these experiments, because the fluorescence of DsbA increases strongly when its disulfide bond is reduced upon reaction with a substrate (Wunderlich and Glockshuber, 1993). Under the chosen conditions the increases in fluorescence and in activity during refolding follow very similar time courses as shown for the oxidized protein in Fig. 4.

0.4 mM GSSG was present throughout to provide oxidizing conditions for DsbA before and during the experiments. The oxidation of C2S/C10N-RNase T1 in the absence of DsbA by 0.4 mM GSSG alone is extremely slow at pH 7.0. It proceeds with a tof about 8 h, and thus GSSG cannot compete with DsbA in the oxidation of C2S/C10N-RNase T1.

Oxidation is a bimolecular reaction between the reduced protein and DsbA, and a second-order rate constant is obtained for this reaction from the analysis by HPLC. To facilitate the comparison between the rate constants for the oxidative formation of the disulfide bond and for the refolding reaction (which is a first-order process), we multiply the second-order rate constant of oxidation with the concentration of DsbA (8 µM) to obtain a rate constant of pseudo first-order. This is clearly an oversimplification, because DsbA is not in vast excess over C2S/C10N-RNase T1 in our experiments and because the reformation of oxidized DsbA by reaction with GSSG occurs only with a rate constant of about 10-20 M s at pH 7.0 and 25 °C (Zapun et al., 1993). Nevertheless, the errors introduced by this conversion are small and do not affect the conclusions that are drawn from this analysis. We give generally time constants for the folding and oxidation reactions. They are the reciprocals of the apparent rate constants.

Oxidation of C2S/C10N-RNase T1 Starting from the Unfolded State

The time course of oxidation of 5 µM C2S/C10N-RNase T1 by 8 µM DsbA at pH 7.0 and 25 °C is shown in Fig. 6A. Prior to this experiment, C2S/C10N-RNase T1 was incubated in 10 mM HCl to maintain it in a reduced and unfolded state (cf. Fig. 1). DsbA is a very efficient oxidant, and the disulfide bond between Cys^6 and Cys is formed rapidly with a time constant of about 20 s, although the two thiol groups are separated by 98 residues. The product of this reoxidation reaction is not the native protein, but an unfolded chain with an intact disulfide bond. The native, catalytically active state is reached in a slow reaction with a time constant of 350 s (Fig. 6A). The kinetics of this reactivation reaction are virtually identical with the refolding kinetics of C2S/C10N-RNase T1 molecules in which the Cys^6-Cys disulfide bond was left intact after unfolding. Apparently, there is a clear order of events in the oxidative folding of C2S/C10N-RNase T1 under the conditions of Fig. 6A. First, the Cys^6-Cys disulfide bond is formed very rapidly by DsbA in the unfolded protein chains, and then these cross-linked molecules refold slowly to the native, catalytically active state.


Figure 6: A, oxidation of unfolded reduced C2S/C10N-RNase T1 by DsbA. Oxidative folding was initiated by a 50-fold dilution of the reduced and unfolded protein (in 10 mM HCl) to final concentrations of 5 µM C2S/C10N-RNase T1, 8 µM oxidized DsbA, and 0.4 mM GSSG in 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. bullet, increase of the fraction of oxidized protein; circle, increase of the fraction of native, enzymatically active protein. The kinetics of reactivation of unfolded molecules with an intact disulfide bond (up triangle) are shown for comparison. They are taken from Fig. 4. The fraction of oxidized protein was measured by reversed-phase HPLC, and reactivation was measured by enzyme assays as described under ``Experimental Procedures.'' The solid curve represents a nonlinear least squares fit of the data for reoxidation to a second-order rate equation and gives a rate constant of oxidation 7 times 10^3 sM. The regain of enzymatic activity is well described by a single first-order reaction with a time constant of 350 s, as indicated by the broken line. B, oxidation of reduced and folded C2S/C10N-RNase T1. The reduced protein was first incubated for 1 h in 2.8 M NaCl, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. At time 0 oxidation was initiated by a dilution to final concentrations of 5 µM C2S/C10N-RNase T1, 8 µM oxidized DsbA, and 0.4 mM GSSG in 2.5 M NaCl, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. bullet, increase of the fraction of oxidized protein; circle, increase of the fraction of native, enzymatically active protein. The solid curve represents a nonlinear least squares fit of the data for oxidation to a second-order rate equation and gives a rate constant for oxidation of 10.4 times 10^3 sM. The regain of enzymatic activity is well described by a single first-order reaction with a time constant of 24 s, as indicated by the broken line.



Oxidation of C2S/C10N-RNase T1 Starting from an Ordered Reduced State

As shown in Fig. 2, an ordered conformation can be generated in reduced C2S/C10N-RNase T1 when the concentration of NaCl is increased above 1.5 M. To examine, whether the rate of formation of the Cys^6-Cys disulfide bond by DsbA depends on the conformation of the reduced protein, we incubated C2S/C10N-RNase T1 first in 2.8 M NaCl, pH 7.0, for 1 h to induce this ordered native-like structure. Then oxidation was started by adding 8 µM DsbA, and the formation of the disulfide bond and the regain of RNase activity were monitored by HPLC and by activity assays, respectively.

In control experiments the rate constant for the reaction of oxidized DsbA with dithiothreitol was measured as a function of the concentration of NaCl at pH 7.0, 25 °C. The rate of this reaction was found to be independent of the NaCl concentration between 0 and 3 M (data not shown), suggesting that the activity of DsbA as an oxidant is not affected by the addition of NaCl.

The kinetics of oxidation of the folded form of reduced C2S/C10N-RNase T1 are shown in Fig. 6B. The ordered structure present after preincubation in 2.8 M NaCl had virtually no effect on the rate of disulfide bond formation; it proceeded with a time constant of about 20 s as in the oxidation of the unfolded form (Fig. 6A). The products of the oxidation reactions in Fig. 6, A and B, were strongly different, however. In the absence of NaCl unfolded cross-linked protein chains were generated by the reaction with DsbA, which then refolded slowly after the disulfide bond had formed. When the reduced protein was preincubated in 2.8 M NaCl, disulfide bond formation and the regain of enzymatic activity followed the same time course. Oxidation by DsbA immediately produced native and catalytically active C2S/C10N-RNase T1.

This indicates that the rank order of folding and oxidation can be reversed by changing the experimental conditions. Either the Cys^6-Cys disulfide is formed first by DsbA in the unfolded protein chains, followed by slow folding, or else, the reduced protein chains can be folded first, followed by the introduction of the disulfide bond into the native-like folded protein. Surprisingly, the Cys^6-Cys disulfide bond is formed by DsbA with very similar rates in the native-like folded and in the unfolded form of C2S/C10N-RNase T1.

Oxidation of C2S/C10N-RNase T1 in the Presence of Prolyl Isomerase

In the next experiment the oxidative folding of reduced and unfolded C2S/C10N-RNase T1 was carried out in the presence of 1000 PU/ml prolyl isomerase. Again, the formation of the disulfide bond by DsbA was a very rapid reaction (Fig. 7A) and showed a time constant of about 15 s, which is slightly faster than the oxidation by DsbA in the absence of prolyl isomerase (cf. Fig. 6A). The subsequent folding reaction to the native state was 6-fold accelerated by prolyl isomerase and showed a time constant of 60 s (Fig. 7A), rather than 350 s, as measured in the absence of prolyl isomerase (Fig. 6A).


Figure 7: A, oxidation of unfolded reduced C2S/C10N-RNase T1 by DsbA in the presence of 1000 PU/ml prolyl isomerase. Oxidation of 5 µM C2S/C10N-RNase T1 by 8 µM oxidized DsbA in 0.4 mM GSSG, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C was carried out and measured as in Fig. 6. bullet, increase of the fraction of oxidized protein; circle, increase of the fraction of native, enzymatically active protein. The kinetics of reactivation in the absence of prolyl isomerase (box and dotted line, taken from Fig. 6A) are shown for comparison. The solid curve represents a nonlinear least squares fit of the data for reoxidation to a second-order rate equation and gives a rate constant of oxidation of 9.4 times 10^3 sM. The regain of enzymatic activity is well described by a single first-order reaction with a time constant of 59 s, as indicated by the dashed line. B, oxidation of reduced C2S/C10N-RNase T1 after an incubation for 1 min of the reduced protein in the presence of 1000 PU/ml prolyl isomerase in 2.8 M NaCl, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. At time 0 oxidation was initiated by a dilution to final concentrations of 5 µM C2S/C10N-RNase T1, 8 µM oxidized DsbA, and 0.4 mM GSSG in 2.5 M NaCl, 0.1 M Hepes, 2 mM EDTA, pH 7.0 at 25 °C. bullet, increase of the fraction of oxidized protein; circle, increase of the fraction of native, enzymatically active protein as a function of oxidation time. The solid curve represents a nonlinear least squares fit of the data for reoxidation to a second-order rate equation and gives a rate constant of oxidation of 12.4 times 10^3 sM. The regain of enzymatic activity is described by a single first-order reaction with a time constant of 19 s, as indicated by the dashed line.



The roles of DsbA and of prolyl isomerase in the oxidative folding of C2S/C10N-RNase T1 can be inferred from a comparison of the results in Fig. 6A and Fig. 7A. In both cases the disulfide bond is formed very rapidly in the unfolded protein by DsbA and then the native structure is formed in the oxidized protein chains. This folding reaction is limited in rate predominantly by the trans cis isomerization of Pro and is catalyzed by prolyl isomerase. The catalysis by prolyl isomerase of the folding of the disulfide-bonded protein is, however, not very efficient.

Oxidation of C2S/C10N-RNase T1 after Catalyzed Chain Folding

The rank order of oxidation and catalyzed refolding can be reversed when DsbA and prolyl isomerase are added at slightly different times. The refolding of reduced C2S/C10N-RNase T1 is catalyzed very efficiently by prolyl isomerase (cf. Fig. 3A), and therefore a short 60-s incubation of the unfolded and reduced protein in the presence of 1000 PU/ml prolyl isomerase and 2.8 M NaCl is sufficient to generate the native-like folded form of the reduced protein with Pro and Pro, both in the cis conformation. When DsbA is added after this short refolding pulse, the native, catalytically active protein with the disulfide bond is formed very rapidly with a time constant of less than 15 s (Fig. 7B).


DISCUSSION

The oxidative folding of C2S/C10N-RNase T1 consists of three major processes: the acquisition of the ordered chain conformation, the formation of the disulfide bond, and the trans cis isomerizations of Pro and Pro. The rank order of these three processes is not fixed, but can differ, depending on the conditions for folding. When DsbA is the oxidant the disulfide bond is formed very rapidly in the unfolded protein, followed by the fast formation of partial structure in the cross-linked molecules. Further folding is then inhibited by the presence of incorrect prolyl isomers, and the slow trans cis isomerizations of Pro and Pro determine the rate of formation of the native, catalytically active protein. These final reactions are accelerated by prolyl isomerase. This particular sequence of events is not dictated by the folding mechanism, but is simply a consequence of the extremely high efficiency of DsbA as an oxidant. It links Cys^6 with Cys in the unfolded protein chains very rapidly, even at pH 7.0.

Such an efficient oxidant is needed, because low molecular weight disulfides, such as GSSG, are almost unreactive at pH 7.0 (Szajewski and Whitesides, 1980). The oxidation of C2S/C10N-RNase T1 by 0.4 mM GSSG proceeds with a t of 7-8 h at pH 7.0, whereas the oxidation by 8 µM DsbA shows a tof about 10 s. The corresponding second-order rate constant for oxidation is 7 times 10^3M s, which is in the range of values reported by Wunderlich et al. (1993a, 1993b) and by Zapun et al.(1993) for the reaction of DsbA with reduced proteins. The oxidized protein chains that are formed by the rapid reaction with DsbA are, however, not good substrates for prolyl isomerase, because partial structure forms rapidly around Pro and decreases its accessibility. The proline-limited folding to the native state therefore remains a slow process, even in the presence of high concentrations of prolyl isomerase.

The native state of C2S/C10N-RNase T1 was reached extremely rapidly when the reduced protein was allowed to fold in the presence of prolyl isomerase for a short period of time (60 s), before DsbA was added. This folding in two steps was very efficient, because prolyl isomerization is much better catalyzed in the reduced protein, and because native-like folded reduced C2S/C10N-RNase T1 is rapidly oxidized by DsbA. It should be noted that a variation in the sequence of folding events was also found in the folding of the beta(2) subunit of tryptophan synthase (Murry-Brelier and Goldberg, 1989).

It is surprising that the Cys^6-Cys disulfide bond is formed by DsbA with almost the same rate in the native-like folded and in the unfolded form of reduced C2S/C10N-RNase T1. In native RNase T1 the Cys^6-Cys disulfide bond is inaccessible to solvent (Heinemann and Saenger, 1982; Martinez-Oyanedel et al., 1991) and is not readily reduced (Pace and Creighton, 1986). Therefore, in the first step of oxidation, the reactivities of Cys^6 and Cys to form a mixed disulfide with a bulky oxidant, such as DsbA, might be decreased when folded structure is present already in the reduced protein. On the other hand, the effective concentration of the thiol groups should be strongly increased in such a native-like folded state, and the intramolecular disulfide exchange reaction in the second step should be accelerated. It is possible that these two effects compensate each other and lead to the observed insensitivity of the overall rate of disulfide bond formation.

The results obtained here and in previous work on the folding of RNase T1 reveal a few aspects that could be important for protein folding in general. (i) During folding, both the isomerization of prolines and the formation (and isomerization) of disulfide bonds have to be linked with the formation of native-like ordered structure. This coupling is necessary to select and to stabilize the correct prolyl isomers and the native disulfide bonding pattern. (ii) A premature formation of ordered structure can have serious adverse effects on the entire folding process. It could lock the protein chains in nonproductive intermediates, it could severely limit the access of prolyl isomerase, and it could block the formation and isomerization of disulfide bonds by thiol-disulfide oxidoreductases, such as DsbA. All these effects can lead to a strong deceleration of folding. Indeed, a nonproductive folding intermediate with incorrect heme ligation was identified for cytochrome c (Ikai et al., 1973; Brems and Stellwagen, 1983; Sosnick et al., 1994; Elöve et al., 1994), a hindrance of prolyl isomerization by premature structure formation was observed in the refolding of RNase T1 (Kiefhaber et al., 1992b), and a strong retardation of thiol/disulfide exchange in folding intermediates was found for pancreatic trypsin inhibitor (Weissman and Kim, 1991; Creighton, 1992, 1994; Zhang and Goldenberg, 1993) and for the immunoglobulin light chain (Goto and Hamaguchi, 1981).

Evidently, intermediates are necessary because they contribute specificity to the folding process, but at the same time there is a high risk that their formation decelerates later steps of folding. This apparent contradiction can be resolved when the stability of folding intermediates remains very low and when they exist in rapid exchange with each other and with the unfolded protein. The stability of the folded structure should increase only in the final rate-limiting step of folding. In other words, folding should be a cooperative process, in which virtually all interactions of the folded state are necessary to stabilize the native conformation. Proteins with such ``ideal'' folding (Go, 1983) are probably exceptional, and the occurrence of nonproductive intermediates might be widespread (Creighton, 1994). In the course of cellular folding, chaperones probably bind to such intermediates and promote their unfolding in an ATP-dependent manner (Todd et al., 1994).

The conditions of our oxidative folding experiments were chosen such that they resemble the conditions in the periplasm of E. coli in some aspects. A protein with a low disulfide density was used as a model, DsbA was the oxidant, and the periplasmic prolyl isomerase was the catalyst of folding. The concentrations of these folding enzymes are not known very well (Akiyama et al., 1992; Hayano et al., 1991); they are presumably higher than those used in this study. Our finding that an ordered sequence of the folding steps is not obligatory for reaching the native state might be relevant for folding in vivo as well. A moderate degeneracy of the folding process should be of advantage and should have been conserved during evolution. This holds in particular for folding in the periplasm, because the solvent conditions in this compartment are not as well controlled as in the cytoplasm or in the endoplasmic reticulum of eukaryotic cells. The general problem that folding intermediates are necessary to direct the folding process, but at the same time restrict the accessibility for the oxidant and for catalysts of folding is relevant for folding in vitro as well as in vivo.


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-921-55-3660; Fax: 49-921-55-3661.

(^1)
The abbreviations used are: RNase T1, ribonuclease T1; C2S/C10N-RNase T1, a variant of ribonuclease T1 with the substitutions Cys^2 Ser and Cys Asn; DsbA, thiol-disulfide oxidoreductase from the periplasm of E. coli; GpC, guanylyl(3`-5`)cytidine; GdmCl, guanidinium chloride; DTE, dithioerythritol; DTT, dithiothreitol; GSH and GSSG, the reduced and oxidized forms of glutathione, respectively; PU, unit for the relative activity of prolyl isomerase. The numerical value of the activity is given by k/k(o) -1, where k(o) is the rate constant of the uncatalyzed isomerization of the peptide Suc-Ala-Ala-Pro-Phe-4-nitroanilide, and k is the rate constant of catalyzed isomerization in the presence of prolyl isomerase; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Matthias Mücke, Thomas Schindler, and Stefan Walter for discussions, Nobuhiro Takahashi for a gift of prolyl isomerase, Rudi Glockshuber and Martina Wunderlich for a gift of DsbA, and Ulrich Hahn for help in mutagenesis experiments.


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