(Received for publication, June 26, 1995; and in revised form, August 8, 1995)
From the
Scrambled hirudins serve as relevant folding intermediates during in vitro renaturation of the reduced/denatured hirudin. Eleven species of scrambled hirudins were isolated and structurally identified. Their properties, including their relative stability and the kinetics of their consolidation (disulfide reshuffling) to attain the native structure, have been investigated and are reported here.
Scrambled structures of cystine-containing proteins are fully oxidized (disulfide formed quantitatively) species that contain at least two non-native disulfide pairings. They were first observed during the renaturation of ribonuclease A (RNase A) performed by Anfinsen and colleagues (Anfinsen et al., 1961; Haber and Anfinsen, 1962; Anfinsen, 1973). When reduced/denatured RNase A was allowed to refold in the presence of a denaturant (8 M urea), 99% of the protein was recovered as inactive scrambled species. Despite the finding (Anfinsen et al., 1961) that they were able to self reorganize to acquire the native conformation, scrambled proteins have been generally regarded as dead-end products of abortive folding and largely ignored by protein chemists.
However, from our recent
refolding experiments performed with hirudin (49 amino acids, 3
disulfides), potato carboxypeptidase inhibitor (PCI) ()(39
amino acids, 3 disulfides), and human epidermal growth factor (53 amino
acids, 3 disulfides), scrambled species have been found to exist along
the folding pathway in all three cases and under a wide range of
folding conditions (Chatrenet and Chang, 1993; Chang et al.,
1994, 1995a). Their foldings undergo an initial stage of nonspecific
disulfide pairing that leads to the formation of scrambled species as
essential intermediates. This is followed by disulfide reshuffling of
scrambled species in the presence of thiol catalyst to reach the native
structure. The accumulation of scrambled species thus can be enhanced
through manipulation of folding conditions by either accelerating the
speed of disulfide formation or halting the process of disulfide
reshuffling. The mechanism of halting disulfide reshuffling has been
demonstrated in the example of folding carried out in the buffer alone.
Under these conditions, free cysteines of 1- and 2-disulfide
intermediates act as thiol catalyst during the early stage of folding.
As the process advances, free cysteines begin to deplete because more
cysteines become involved in disulfide pairing and less are available
as thiol catalyst for disulfide reshuffling, therefore scrambled
species become trapped and accumulated in an exponential manner. By
this method, about 50-60% of the protein was consistently
recovered as stable scrambled species (Chang, 1994). Alternatively, a
high yield of scrambled species could be obtained by promoting the
efficiency of disulfide formation. For instance, when folding of PCI
was performed in the presence of 0.5 mM of cystine, more than
99% of the total protein was trapped as scrambled species before trace
amounts of the native PCI even appeared (Chang et al., 1994).
The reproducible appearance of scrambled species in such significant concentrations indicates that they could not be simply dismissed as by-products or abortive structures of ``off-pathway'' folding. As a matter of fact, they have also been observed during the productive folding of ribonuclease A (Creighton, 1979) and pro-BPTI (Weissman and Kim, 1992) that involved no denaturants. In our opinion, scrambled species represent logical intermediates in a folding process that is governed by thermodynamic principles (Anfinsen, 1973). Their conversion to the native structure, which is apparently driven and guided by noncovalent specific interactions, accounts for the major event of protein folding. There are 14 possible scrambled species for a protein containing three disulfides. In the case of hirudin, 11 of them have been isolated and structurally identified (Chang et al., 1995b). In this report, we investigate their relative stability and the kinetics of their conversion to attain the native structure.
Figure 1: End products of hirudin folding carried out in the alkaline buffer alone. About 60-65% of the protein was trapped as scrambled species, unable to convert spontaneously to the native structure due to the absence of free thiols as catalyst. Ten fractions of scrambled hirudins were identified, and their disulfide structures have been characterized. Fraction c contains two scrambled species with approximately equal concentration. The sample was analyzed by HPLC using the following conditions. Solvent A was water containing 0.1% trifluoroacetic acid. Solvent B was acetonitrile/water (9:1, v/v) containing 0.1% trifluoroacetic acid. The gradient was 14-32% solvent B linear in 50 min. The column was Vydac C-18 for peptides and proteins (4.6 mm, 10 µm). Column temperature was 22 °C.
For enzymatic digestion, hirudins (100 µM) were incubated at 22 °C with Glu-C protease (20:1 or 10:1, substrate/enzyme, w/w) in the ammonium bicarbonate buffer (50 mM, pH, 8.0). Aliquots of the digest were removed in a time-course manner, and amino termini generated by the digestion were quantitatively evaluated by the dimethylaminoazobenzene isothiocyanate method (Chang, 1988).
This state of equilibrium is perturbed in the presence of a
denaturant. Denaturants display two important effects. First, they
alter the equilibrium constant among scrambled species. For example,
the concentrations of fractions a, e, f, g,
and h (as compared with those of fractions a*, b, b*, c, and d) are diminished by 5-fold in the
presence of 6 M GdmCl (Fig. 2). The extent of their
decrease is directly related to the concentration of denaturant.
Second, the presence of denaturant significantly narrows the free
energy gap between the native and scrambled structures. In the presence
of 4, 6, and 8 M GdmCl, the equilibrium constant of scrambled
hirudins/native hirudin increases from <0.001 to 0.30, 1.45, and
5.25, respectively (Fig. 2). For instance, when the native
hirudin was incubated in the Tris-HCl buffer containing 6 M GdmCl and -mercaptoethanol (0.25 mM), 60 ± 2%
of the sample reshuffled its native disulfides and settled at the
scrambled structures. Conversely, when the reduced/denatured hirudin
was allowed to refold in the same solution, only 40 ± 2% of the
protein eventually converted to the native structure (Fig. 3).
The same equilibrium could be obtained by permitting scrambled hirudins
(mixture) to consolidate in the same GdmCl solution. Along this
process, scrambled hirudins reshuffle among themselves to establish a
new state of equilibrium that is accompanied by a drastic decrease of
the denaturant-sensitive species (a, e, f, g, and h) (Fig. 4).
Figure 2:
The
equilibrium constant of scrambled hirudins/native hirudin is dependent
upon the concentration of denaturant. Native hirudin (1 mg/ml) was
incubated in the Tris-HCl buffer containing 0.25 mM -mercaptoethanol and various concentrations of GdmCl. Control
sample was dissolved in the same buffer containing 0.25 mM
-mercaptoethanol without GdmCl. The reaction was allowed for 16 h.
Samples were trapped by acid and analyzed by HPLC. End products contain
a mixture of equilibrated scrambled/native (N) species. The
equilibrium constant (K
) obtained is highly
reproducible with an average deviation smaller than 5%. The inset provides the equilibrium constants. The open arrow indicates the elution position of the fully reduced species.
Another set of experiment demonstrated that the disulfide structure of
native hirudin was not disrupted at all by GdmCl in the absence of
-mercaptoethanol.
Figure 3:
In the presence of 6 M GdmCl,
scrambled hirudins and the native hirudin exist in equilibrium (K 1.45), regardless of whether the equilibration
is driven from the end of native hirudin (unfolding) or the end of
fully reduced/denatured species (R, refolding). Both folding
and unfolding were performed at 22 °C in the Tris-HCl buffer (0.1 M, pH 8.5) containing 6 M GdmCl and 0.25 mM
-mercaptoethanol.
Figure 4:
Consolidation of scrambled hirudins in the
absence (A) and the presence (B) of 6 M GdmCl. The reactions were performed in the Tris-HCl buffer
containing 0.25 mM of -mercaptoethanol as catalyst. Only
38% of scrambled hirudins was able to convert to the native structure
in the presence of 6 M GdmCl.
Similar studies were also conducted in the presence of varied concentrations of urea. The results demonstrate that urea is about 4-fold less potent in denaturing the native hirudin. In the presence of 8 M urea, the equilibrium constant of scrambled/native hirudins is 0.11 ± 0.01.
When an isolated
species of scrambled hirudin was allowed to reshuffle disulfides and
consolidate, it advances simultaneously toward two directions. It
equilibrates with the native structure as well as with other scrambled
species (Fig. 5). Because the equilibration predominantly favors
the native structure, in the end all scrambled species convert
spontaneously to the native hirudin. These reactions were carefully
analyzed by using either -mercaptoethanol, cysteine, or reduced
glutathione as thiol catalyst. The kinetics and mechanisms involved in
those reactions are enormously complicated, but a number of crucial
observations can be concluded from these studies ( Fig. 5and Fig. 6). 1) The initial rate to equilibrate with other scrambled
species is about 4-5 times faster than that of converting to the
native species. This was observed with the time-course analysis of all
11 different scrambled species (Fig. 6). 2) An isolated
scrambled species equilibrates faster with those that already share a
common disulfide bridge. An obvious case is species b*, which
converted to a* about 6-fold faster than to the remaining
scrambled species (Fig. 6). a* and b* happen to be
the only two species that share the largest disulfide loop
Cys
-Cys
(Fig. 1). Another example is
the rapid interconversion of species h and g, both of
which contain Cys
-Cys
. 3) The
denaturant-sensitive species (a, e, f, g,
and h) are relatively less productive in converting to the
native structure. This may in part reflect their better stability,
which is also consistent with the finding that denaturant-sensitive
species are more resistant to reduction (see Fig. 8). With Cys
(1 mM) as catalyst, the most and least productive species
display comparable rate constants of 0.072 (fraction b) and
0.038 min
(fraction f), respectively. Using
reduced glutathione (1 mM), the rate of consolidation is
reduced by half to 0.035 (b) and 0.022 min
(f). In the presence of
-mercaptoethanol (0.25
mM), the rate constants range from 0.016 (b) to 0.01
min
(f).
Figure 5: Consolidation of an isolated scrambled hirudin to acquire the native structure. Fraction d was reconstituted in the Tris-HCl buffer containing 1 mM of reduced glutathione as catalyst. Intermediates during the course of disulfide reshuffling were trapped by acid and analyzed by HPLC using the same conditions described in the legend for Fig. 1.
Figure 6: The intermediates (trapped at 5 min) during the course of consolidation of isolated scrambled hirudins. Cys (1 mM) was used as thiol catalyst in these experiments. The intermediates were trapped by acid. Values given on the left-hand side of the panel indicate the recoveries of native hirudin.
Figure 8: Reduction of scrambled hirudins. The reduction of scrambled hirudins was performed in the presence of 0.2 mM of dithiothreitol. R and N stand for fully reduced and the native hirudins, respectively. The elution positions of the scrambled species (III), 2-disulfide (II), and 1-disulfide (I) species are indicated at the 0-time and 60 min samples. The gradient systems of HPLC are as described in the legend for Fig. 1, except that a 5-µm instead of a 10-µm Vydac C-18 column was used here. With the 5 µm column, fractions e and d are baseline separated, but a* and b* co-elute with b and c, respectively.
The striking outcome is that all fractions of scrambled hirudins behave in a very similar manner. This includes the efficiency to generate the native structure and the rate to reach equilibrium with other scrambled species ( Fig. 5and Fig. 6). No scrambled species has been shown to be particularly productive in generating the native structure. These findings unambiguously demonstrate that scrambled hirudins consist of an assemblage of equilibrated isomers possessing a similar state of free energy.
Figure 7: Recoveries of fully reduced hirudin obtained from the reduction of the native and scrambled hirudins. Conditions of reduction are described in the text, and concentrations of dithiothreitol employed are indicated along with each curve. A, scrambled hirudins. B, the native hirudin. C, the native hirudin in the presence of 6 M GdmCl. In the case of C, the fully reduced hirudin eventually swung back to the 1- and 2-disulfide species due to the oxidation of dithiothreitol.
The results of HPLC analysis reveal the small differences of stability among scrambled species. During the reduction, the decrease of species c and d is about 2-fold faster than that of denaturant-sensitive species e, f, g, and h (a, a*, b, and b* could not be measured here because they overlap with 2-disulfide species) (Fig. 8). Also, species f appears to be slightly more resistant against reduction than e, g, and h. All these are consistent with the sluggish behavior of denaturant-sensitive species during the process of consolidation (Fig. 6). Furthermore, 2- and 1-disulfide species were detected during the reduction of scrambled hirudins (Fig. 8). By contrast, reduction of the native disulfides adopts an ``all-or-none'' (collapse) pathway in which only 1-2% of 1- and 2-disulfide intermediates appear, a mechanism similar to that found during the reductive unfolding of RNase (Creighton, 1979) and observed at dithiothreitol concentrations ranging from 10 to 100 mM.
Enzymatic susceptibility of a protein is commonly linked to the state of its conformation. Compactly folded proteins are usually resistant to enzyme digestion, and most denatured or reduced/carboxymethylated proteins are freely digestible by enzymes. The data obtained here indicate that scrambled hirudins adopt loose conformations that closely resemble the denatured state. (Dill and Shortle, 1991).
Scrambled species are landmark intermediates that permit identification of the two-stage folding mechanism for both hirudin and PCI (Chang et al., 1994). Their formation is a consequence of the nonspecific packing (disulfide pairing) that occurred during the early stage of folding. This two-stage mechanism is compatible with the folding behavior observed with RNase A (Anfinsen et al., 1961; Pigiet and Shuster, 1986). It is also consistent with a recent example of chymotrypsin inhibitor 2 (Jackson and Fresht, 1991; Jackson et al., 1993; Otzen et al., 1994). Chymotrypsin inhibitor 2 is an interesting case because it has a size similar to hirudin but contains no disulfide. A collapsed state of chymotrypsin inhibitor 2 serves as folding intermediate, and its conversion to the native structure also represents the rate-limiting step. In our opinion, the property of this collapsed state of chymotrypsin inhibitor 2 resembles that of scrambled hirudins.
In this study, it has been demonstrated that scrambled hirudins consist of a collection of equilibrated isomers possessing a similar state of free energy. They also exhibit a markedly reduced stability as compared with that of native hirudin, a property that is consistent with what has been described for the ``molten globe'' state (Ptitsyn, 1987; Kuwajima, 1989). There are, however, detectable differences of stability among scrambled hirudins. About half of scrambled hirudins (a, e, f, g, and h) are sensitive to denaturant. They are also less productive in converting to the native structure and more resistant against reduction as compared with species a*, b, b*, c, and d. All this implies that denaturant-sensitive species are stabilized by noncovalent interactions that are partially abrogated in the presence of denaturants. These noncovalent interactions do not necessarily resemble those that fix the native structure, and their precise nature remains to be elucidated by NMR. Interestingly, most of the denaturant-sensitive species (f, g, and h) do not even contain a native disulfide, and the two well-populated species (b and c) that do contain a native disulfide are entirely insensitive to denaturants.
From the viewpoint of solely kinetic
analysis of disulfide formation, one can correctly argue that scrambled
species are dead-end products, because conversion of scrambled species
to the native structure must undergo disulfide reshuffling and, in
practice, 2-disulfide or even 1-disulfide species must serve as
transitory intermediates. However, this argument misses the point that
formation of disulfides are consequences, not causes, of folding and
that they are used as signals to trace, not to dictate, the mechanism
of folding. Thermodynamically, the presence of scrambled structures as
folding intermediates is perfectly logical. Scrambled species simply
represent a state of more advanced packing and relatively lower free
energy (G) than that of 2-disulfide species. In this
case, the loss of entropy (randomness) (
S) is more than
compensated by the decrease of enthalpy (
H), due to the
reordering of the hydrophobic structural elements in the aqueous
environment. This proposal is supported by two important observations:
1) the flow of 2-disulfide intermediates to the scrambled species is
spontaneous and irreversible during the folding, and 2) scrambled
species are eluted earlier than 2-disulfide and 1-disulfide species on
reverse phase HPLC (Chatrenet and Chang, 1993). Their conversion to the
native structure, although accompanied by the disulfide reshuffling,
does not necessarily require unfolding of the compact structure they
have already attained. Indeed, during the process of conversion from
scrambled species to the native structure, 2-disulfide and 1-disulfide
intermediates have not been observed (Chang, 1994); clearly they must
exist in equilibrium in such a minute concentration that it is beyond
detection.
The presence of scrambled species as folding
intermediates features a major distinction between the folding
mechanism of the BPTI (Creighton, 1978, 1990; Weissman and Kim, 1991)
and hirudin (Chatrenet and Chang, 1993) models. Similar to hirudin,
there are species of folding intermediates of BPTI that become trapped
during the folding and are unable to convert to the native structure
(Weissman and Kim, 1991, 1992). In hirudin, they are identified as
3-disulfide scrambled species, and their trapping is due to the absence
of thiol reagent to catalyze their further disulfide reshuffling. In
the case of BPTI, these trapped intermediates have been characterized
as 2-disulfide (native) species with buried cysteines that are
incapable or extremely slow to form the third native disulfide. Aside
from scrambled species, some conceptual differences concerning the
definition of a folding pathway also need to be clarified. The crucial
question is what types of folding intermediates actually constitute the
pathway, the well populated intermediates or the productive species
that account for the flow of folding? Also, how are well populated
intermediates quantitatively defined? Should one ignore those minor
intermediates that constitute less than 10-15% of the well
populated species? These are important issues because well populated
species are not necessarily the productive ones when isomers of
intermediates exist in a state of dynamic equilibrium as those observed
in the cases of hirudin and BPTI. Should well populated intermediates
be chosen to specify the pathway, then it is obvious that hirudin, PCI,
and RNase (Scheraga et al., 1984, 1987) all fold via multiple
pathways because of the heterogeneity of their well populated
intermediates. Even in the case of BPTI, the intermediates are probably
far more complicated than what has been described (Weissman and Kim,
1991). Using HPLC and mass spectrometry, we have detected more than 46
fractions of folding intermediates of BPTI, ()although some
of the minor fractions represent only 10-20% of the predominant
species. If, on the other hand, one agrees that the productive
intermediates specify the pathway, then those productive species still
remain to be identified in the cases of hirudin, PCI, and RNase, as
well as BPTI. We suggest that one way to identify the productive
intermediates is through kinetic analysis of purified, structurally
defined intermediates (both major and minor) using the stop/go folding
experiments (Chang, 1993). With a protein containing three disulfides,
this approach is facing two formidable challenges. The first is to
separate and isolate all intermediates, major or minor, that are
present at the same stage of equilibrium to homogeneity. Considering
the possible isomers that exist (15 1-disulfide and 45 2-disulfide
species) and the apparent complexity of intermediates observed with
hirudin and PCI, this will be a daunting task. The second challenge,
which is even more intractable, is to ensure that all minor species
have been found and isolated, including those that exist with
concentrations that may be less than 1% of the well populated species.
This work is dedicated to the author's late father, Tschenda Chang.