(Received for publication, November 10, 1994; and in revised form, December 19, 1994)
From the
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 2 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
10
s
M
), 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.
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) ()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-Cys
and
Cys
-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
-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
2 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
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.
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.
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-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-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
-Cys
and Cys
-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.
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
-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
() and by the regain of enzymatic activity (
) 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.
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.
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. , increase
of the fraction of oxidized protein;
, increase of the fraction of
native, enzymatically active protein. The kinetics of reactivation of
unfolded molecules with an intact disulfide bond (
) 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
10
s
M
. 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.
,
increase of the fraction of oxidized protein;
, 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
10
s
M
. 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.
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-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
-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.
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. , increase of the fraction of
oxidized protein;
, increase of the fraction of native,
enzymatically active protein. The kinetics of reactivation in the
absence of prolyl isomerase (
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
10
s
M
. 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.
,
increase of the fraction of oxidized protein;
, 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
10
s
M
. 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.
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
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 t
of
about 10 s. The corresponding second-order rate constant for oxidation
is 7
10
M
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 subunit of tryptophan synthase
(Murry-Brelier and Goldberg, 1989).
It is surprising that the
Cys-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
-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
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.