(Received for publication, October 1, 1995; and in revised form, February 15, 1996)
From the
Oxidative folding of recombinant human stem cell factor (rhSCF)
produced in Escherichia coli was investigated in
vitro. Folding of denatured and reduced rhSCF involves at least
five intermediate forms, I-1 to I-5, detectable by their differences in
hydrophobicity using reverse-phase high performance liquid
chromatography. Both I-1 and I-2 contain a native-like disulfide bond,
Cys-Cys
and
Cys
-Cys
, respectively, and I-3 forms a
mispaired disulfide, Cys
-Cys
. These
forms appear to reach steady state equilibrium and are important
folding intermediates. I-1 was found to be the prominent intermediate
that directly folds into native rhSCF(N); and the thermodynamically
less stable I-2 favors rearrangment into I-1. I-3 may serve as an
intermediate for disulfide rearrangement between I-1 and I-2. I-4 and
I-5, which are disulfide-linked dimers, are in equilibrium with reduced
rhSCF and other intermediates and may not play an important role in
rhSCF folding. Both trifluoroacetic acid-trapped I-1 and I-2, after
isolation by high performance liquid chromatography, proceed with the
remaining oxidative folding process after reconstitution.
Iodoacetate-trapped I-1 and I-2 contain low
-helical content and
some tertiary structure, while I-3 and reduced rhSCF have little
ordered structure. Gel filtration/light-scattering experiments indicate
that reduced rhSCF and iodoacetate-trapped I-1, I-2, and I-3 exist as
dimeric forms, indicating that rhSCF dimerization precedes formation of
disulfide bonds. I-1, I-2, I-3, and the C43,138A analog lacking
Cys
-Cys
bond are not biologically
active or exhibit significantly lower activity. The two disulfide bonds
in rhSCF seem to be essential for the molecule to maintain an active
conformation required for its receptor binding and biological
activities.
The mechanistic folding of proteins is a critical area of study in understanding their structural and conformational behaviors in solution. Much experimental work has been done on the thermodynamics and kinetics of folding pathways of proteins(1, 2, 3, 4) . Disulfide-containing proteins have the advantages that the intra- or interchain disulfide bond is a natural covalent cross-link which, by thermodynamic requirements, stabilizes the cross-linked conformation (1) . Oxidative refolding of a disulfide-reduced protein allows one to study the rates of folding and to chemically trap the folding intermediates. Many such folding intermediates are sufficiently long-lived during folding and can be isolated and characterized. In several cases, detailed folding pathways have been defined in terms of occurrence of disulfide-containing intermediates at various stages of folding(4, 5, 6, 7) . Through the analysis of disulfide intermediates, it is apparent that disulfide formation does not necessarily occur through a simple sequential pathway. As a result, specific issues regarding protein folding pathways have been brought to attention(3, 6, 7, 8, 9, 10) . These include the significance of non-native disulfides, the kinetic importance of well populated and thermodynamically stable intermediates, and the clarification of the consequence and role of kinetically trapped species that accumulate in the pathway.
Human
stem cell factor (hSCF) ()is a hematopoietic cytokine that
plays an important role in the stimulation, proliferation,
differentiation, and functional activation of blood
cells(11, 12) . The molecules obtained naturally or
expressed in recombinant CHO cells are heavily
glycosylated(13, 14, 15) . Recombinant hSCF
(rhSCF) can also be produced in genetically engineered Escherichia
coli(16) . The glycosylated or nonglycosylated molecule
exists as a noncovalently linked homodimer in solution(17) .
Dissociation-reassociation of rhSCF dimer spontaneously occurs at a
fairly rapid rate(18) . There are two intramolecular disulfide
bonds present in the molecule (Cys
-Cys
and Cys
-Cys
), and the production
of active rhSCF from E. coli requires an oxidative folding
procedure to recover its biological activity(16) . In this
study, the refolding pathway of reduced rhSCF is examined by monitoring
the distribution of HPLC-separable disulfide-bonded intermediates
during refolding and by characterizing physicochemical and biological
properties of the isolated intermediates. The folding of rhSCF involves
intermediates containing native and non-native disulfides,
thermodynamically stable and unstable intermediates, as well as
disulfide-bonded dimers. These studies allow us better to understand
the contribution of native and mispaired disulfide-containing
intermediates to the in vitro folding pathway of rhSCF.
The
refolding rhSCF mixtures, after IAA or acid treatment, were injected
onto C-4 reverse phase columns (Syn-Chro Pak, 0.46 25 cm, 300
Å). Solvent A is 0.1% TFA and solvent B is 0.1% TFA in 90%
acetonitrile. The column was initially equilibrated with 55% B and
eluted by linear gradients from 55 to 65% B over 80 min and from 65 to
95% B over 5 min on an HP-1090 liquid chromatograph equipped with an
autosampler and a photodiode array detector. Elution of protein peaks
was detected at 215 nm, and protein fractions obtained from HPLC were
collected for further analysis.
The fluorescence spectra were determined at ambient temperature on an SLM Aminco 500C spectrofluorimeter, using a cuvette with a path length of 0.5 cm and protein concentrations of 0.22 mg/ml. The solutions were excited at 280 nm, and the fluorescence spectra from 280 to 420 nm were recorded, using slit widths of 5 nm.
Figure 1: HPLC analysis and kinetics of rhSCF folding products. RP-HPLC of rhSCF intermediates after iodoacetic acid reaction prepared at 1 mg/ml (panel A) and folding kinetics for the disappearance of reduced rhSCF (R) and the appearance of the fully folded rhSCF (N) and partially folded intermediates (I-1 to I-5) over a period of 50 h and 20 min (panels B and C, respectively). The percent generation of rhSCF forms represents peak area of each intermediate divided by the sum of the peak area of all intermediates at the given time.
Fig. 1A shows RP-HPLC analysis of IAA-treated sample aliquots taken at different time intervals. Folding after TFA acidification also exhibited a similar HPLC profile containing the same relative distribution of intermediate forms. RP-HPLC can resolve several IAA-trapped intermediates in addition to the native(N) and fully reduced (R) forms in rhSCF folding mixtures. In the initial stages of the reaction (<2 h), R and N species were readily identified along with five intermediate forms, designated as I-1, I-2, I-3, I-4, and I-5. I-4 and I-5 seemed to form very early (<20 min) and decreased to a very low level later. Intermediate I-1, I-2, and I-3 still remain to some extent throughout the whole folding process. At later stages in the folding (>20 h) a particular form P that elutes more hydrophobically than R also appeared. P was identified to be an SDS-nondissociable but DTT-reducible rhSCF dimer as described in a companion study(26) .
The folding kinetics were investigated further by plotting folding times versus rate of generation for each intermediate estimated from HPLC peak integration. Fig. 1B shows the kinetic profiles for the five intermediate forms as well as R and N, while Fig. 1C depicts generation rates for intermediates within the first 20 min of folding. Initially R disappeared rapidly with concomitant formation of various intermediate forms and the finally folded form N. The data clearly show that the I-1 concentration increased rapidly and persisted for a longer period of time during folding. After 48 h of folding, 72.9% of the total protein has completely folded into N, with 12.1% remaining in I-1 and 12.9% in P. Only 1.3% of the protein remained fully reduced.
The initial first order folding rates for conversion of R into each of the intermediates and the time required to reach maximal concentration derived from the results shown in Fig. 1are listed in Table 1. Initial rates of I-1, I-2, and I-3 formation are similar. However, the time required for I-1 to reach its higher maximal concentration is much longer than those for I-2 and I-3. I-4 and I-5 reached maximal concentration by two min after which their rates of formation decreased. As a result, their initial rate constants could not be determined. In separate folding experiments, decreasing rhSCF concentration (i.e. 0.43 mg/ml) has dramatically reduced the initial folding rate and increased the times for intermediates to reach their maximal concentrations (Table 1). For, example, the initial folding rate has decreased 5 to 10-fold for I-1, I-2, and I-3. In the folding at lower rhSCF concentrations (0.2-0.5 mg/ml), the disulfide linked dimer, I-4, and I-5, have diminished.
Different rhSCF intermediates were subjected to nonreducing SDS-PAGE. As listed in Table 2, R migrates as an extended, larger molecule than the native (18 and 15 kDa, respectively). IAA-trapped I-1 and I-3 also have expanded molecular sizes between N and R. Both I-4 and I-5 migrated as a dimer having a molecular mass of 38 kDa. Upon reduction, their molecular masses reduced to 18 kDa.
Figure 2:
Peptide mapping of alkylated intermediates
after endoproteinase Glu-C digestion. Chromatograms 1-4,
R, I-3, I-1, and I-2, respectively, separated on a C4 column (inside
diameter = 0.46 25 cm). Chromatograms
5-7, R, I-4, and I-5, respectively, separated on a narrow
bore C4 column (0.21
25 cm). The assigned Cys(Cm)- and
disulfide-containing peptides are
identified.
Figure 3: Analysis of folding and oxidation of isolated acid-trapped intermediates by RP-HPLC. Panels A and B, folding of I-1 and I-2, respectively. Generation of fully fully folded (N), reduced (R), and intermediates are identified. X is not characterized.
Figure 4:
Refolding kinetics for rhSCF modified
forms. Panel A, percent regeneration represents the formation
of Cys-Cys
from folding of rhSCF
C43,138A analog (
-
) which is compared to the sum
of the formation for both I-1 and N (
-
) as
well as I-1 alone (
-
) from reduced rhSCF. Panel B, percent regeneration represents formation of
Cys
-Cys
bond
(
-
) from refolding of iodoacetate-modified
I-2, which is compared to the formation of both I-2
(
-
) and the sum of both I-2 and N
(
-
) from R are also
indicated.
Figure 5: Spectroscopic properties of rhSCF and intermediates. Far UV CD spectra of rhSCF species (panel A), near UV CD spectra of rhSCF species (panel B), temperature-dependent changes in ellipticity at 222 nm for rhSCF species (panel C), and fluorescence spectra of rhSCF species (panel D).
Fig. 5B shows the near UV CD spectra. N also has a spectrum identical in shape to that of standard rhSCF, with a slight decrease in magnitude. The spectrum of R indicates that the reduced protein is structurally disordered. I-1 and I-2 have identical spectra that are also similar in shape to that of N, but with signals reduced at levels between N and R. Both of the single disulfide intermediates appear to partially display a native-like tertiary structure.
The changes in ellipticity at 222
nm versus temperature for various rhSCF forms are depicted in Fig. 5C. Increase of temperature causes loss of
-helical structure as a result of thermal denaturation. Table 2lists the transition midpoint of thermal denaturation of
various rhSCF intermediates and analog. SCF standard and fully refolded
rhSCF(N) are very stable molecules, with identical transition
temperature of 60 °C. I-1 and C43,138A analog with a temperature of
48 °C are of intermediate stability. I-2 with a temperature of 36
°C is the least stable, consistent with increased flexibility as
indicated by its fluorescence spectrum (described below).
The
fluorescence spectra of rhSCF species are shown in Fig. 5D. The spectra for both refolded rhSCF and rhSCF
standard consist of a single peak at 322 nm, consistent with the single
Trp (at position 44) being in a hydrophobic environment. The FWHM is
about 50 nm. As is usually the case for Trp-containing proteins, there
is no Tyr signal, indicating energy transfer is occurring between Tyr
and Trp. R has a single peak at 350 nm with a wider FWHM of 56 nm,
typical of a Trp spectrum in solution; and its Tyr fluorescence is not
apparent. I-1 has a spectrum slightly red shifted to 329 nm, indicating
that the Trp is more exposed to the solvent than N. However, Trp in I-1
appears to be in a native-like environment. I-2 has a fluorescence
maximum at 333 nm with a FWHM of 57 nm, indicating that the Trp in this
molecule is slightly more exposed to solvent than I-1. Like R, the
spectrum of I-3 has a maximum at 350 nm, apparently due to Trp being
fully exposed. I-3 also contains a clear shoulder representing the
presence of substantial Tyr fluorescence, indicating that the Tyr is no
longer transferring energy to the Trp. This suggests that the distance
between the Tyr and Trp has increased. C43,138A analog lacking the
Cys-Cys
disulfide bond, exhibits both
CD and fluorescence spectra identical to those for the rhSCF standard.
Its thermostability, however, is similar to I-1 (see above).
Hydrodynamic properties of several intermediates and analog were
analyzed by size exclusion chromatography in conjunction with
light-scattering detection. As expected, the determined molecular
weights for rhSCF standard, N and intermediates are all around
33,000-35,000, indicative of being a dimer in solution (Table 2). Fig. 6A illustrates a typical
chromatographic separation profile for I-2 detected by UV absorption,
refractive index, and light scattering. Fig. 6B shows
the calibration plot for the molecular weight determination of various
species. N was determined to have a molecular weight close to that of
the rhSCF standard. IAA-trapped I-1, I-3, and I-2 are also dimeric
molecules. With the exception of standard rhSCF and N, every isolated
intermediate and R contain small amounts of high molecular weight
aggregates (only detectable by light scattering; see Fig. 6A for I-2), indicating that intermediates and R are less stable in
solution (in an order of N I-1 < I-2, I-3 < R).
Figure 6: Gel filtration of rhSCF and intermediates. Left panel, elution profiles of I-2 detected by UV absorption at 280 nm, refractive index [RI], and the 90° light scattering [LS] (chromatograms a-c, respectively). The column was calibrated with standard proteins as described in the right panel. The main peak eluting at 15 min was detected all methods and identified to be the dimeric SCF. The early eluting peak in chromatogram c, possibly the aggregated tetrameric I-2, can only be detected by light scattering. Right panel, molecular weight determination of rhSCF forms. [LS]/[RI] values were plotted versus molecular weight of standard proteins as described under ``Experimental Procedures.'' BSA, bovine serum albumin.
In vitro folding of rhSCF proceeds via a number of intermediate forms containing native and non-native disulfide bonds. There are five partially oxidized intermediates together with reduced rhSCF. At the completion, the folding mixture contained a major folded and oxidized form N along with small fractions of I-1 and P. I-1, I-2, and I-3 appear to reach steady state equilibrium during folding (Fig. 1), implying that they are important intermediates in the rhSCF folding pathway. From refolding studies of the isolated intermediates and analog ( Fig. 3and Fig. 4), it appears that I-1 is an essential intermediate directly folding into N, while the majority of I-2 needs to reconvert to I-1 via I-3 by S-S rearrangement. By estimation of equilibrium concentrations detected by HPLC (Table 1), I-1 is the most stable intermediate while I-2 and I-3 are less stable.
Several observations indicated that the
conformation of I-1 is mostly native-like. For example, I-1 is the only
intermediate still retaining residual biological activity (5%) (Table 3). Upon SV-8 proteolytic digestion, an appreciable amount
of I-1 remains as large, undigested fragments, whereas other
intermediates can be easily digested (Fig. 2). By fluorescence
spectroscopic analysis, Trp in I-1 or I-2 appears to be in
a hydrophobic environment similar to that found for rhSCF
standard(17) . By partial reduction of native rhSCF, I-2 is the
major species accumulated; and therefore stability of
Cys
-Cys
bond is higher than
Cys
-Cys
bond (see (26) for
details). Taken together with the single Trp being in a hydrophobic
core structure, it is highly possible that
Cys
-Cys
is stabilized by being in this
environment. This hydrophobic environment should also stabilize I-1 by
sequestering the free Cys
and Cys
away from
the aqueous surrounding needed for the formation of
Cys
-Cys
bond. As a result, I-1 appears
to be stable and remains as the major intermediate form.
Our data
indicated that the disulfide-mispaired I-3 may serve as an intermediate
for the conversion between I-1 and I-2. This is supported by refolding
of isolated I-2 which converts to I-1 through I-3 by disulfide
rearrangement (Fig. 3B and Fig. 4). Formation of
Cys-Cys
disulfide bond does not
necessarily require the rearrangement since rhSCF C43,138A analog, a
homolog of I-1, forms Cys
-Cys
bond at
the same rate as that in the wild type molecule. However, during
folding of the reduced rhSCF, disulfide rearrangement between I-1 and
I-2 via I-3 actually decreased the folding rate for I-2 (Fig. 4B). The folding of isolated I-2 produced I-1 and
I-3 before N, indicating that the formation of N from I-2 is
preferentially directed to I-1 via disulfide rearrangement (Fig. 3B). Since a small fraction of N was detected in
addition to I-1 and I-3 at the early folding stage of isolated I-2,
direct folding of I-2 into N may also occur at a slow rate.
The
generation of intermolecular disulfide bonds found in I-4 and I-5 is
interesting. They are relatively unstable as they only transiently
exist at an early stage of folding. In the refolding of I-4 and I-5, it
appears that both intermediates refold back into R which then fold into
N via I-1, I-2, and I-3. Since dimerization of either I-4 or I-5 uses
the same cysteine residue (Cys or Cys
,
respectively), their structures may be different from any of the
isolated intermediates as well as product P found at the end of folding
(see subsequent paper, ref. 26). These observations suggest that I-4
and I-5 may not be as important as other intermediates in rhSCF
folding. In summary, properties of all characterized intermediates
appear to indicate that I-1, I-2, and I-3 are on-pathway folding
intermediates important in the folding of rhSCF, while I-4 and I-5 may
be off-pathway intermediates.
In comparison with native rhSCF, each trapped intermediate and R appears to be unstable in solution as they form a large amount of high molecular weight aggregates detectable in gel permeation chromatography using light-scattering detection. This observation implies that rhSCF disulfide bonds and/or the fully folded conformation play a role in the stability of rhSCF molecule. Characterization of partially oxidized intermediates seems to indicate that each of the two rhSCF disulfide bonds may contribute equally in maintaining proper tertiary structural folding of rhSCF.
All rhSCF
folding species exist as noncovalently linked dimers (Table 3).
We observe that the completely reduced rhSCF (R) is also dimeric.
However, the completely denatured and reduced R requires 1-2 h to
become dimeric in oxygen-free folding conditions, and R prepared from
partial reduction experiments in the absence of 6 M GdnHCl is
present in dimeric form as well. ()Refolding rate of this
reduced rhSCF preparation is similar to that for the reduced rhSCF
prepared in 6 M GdnHCl. Therefore, the formation of dimer may
occur at an earlier folding time preceding oxidation of disulfide bonds
and may not affect the overall folding.
To elicit its full
biological function, rhSCF has to be fully oxidized and folded. None of
the isolated intermediates and analog show significant biological
activity, indicating that they do not have native rhSCF conformations.
Cys-Cys
and
Cys
-Cys
are essential to maintain a
proper structural folding of rhSCF for binding to its receptor,
c-kit, and exerting full biological functions. Although the
completely folded and purified C43,138A analog has a gross conformation
indistinguishable from the wild type rhSCF and displays significant
receptor binding activity, this molecule is biologically less active (Table 3).
In summary, a putative in vitro folding
pathway was postulated. I-1 is identified as an important and
productive intermediate for formation of fully oxidized and folded
rhSCF. I-2 seems to favor rearrangment through I-3 to I-1 and N. I-2
may also directly fold into N at a much slower rate. Our preliminary
data indicated that the folding of rhSCF can be affected by protein
disulfide isomerase. It would be interesting to further
study the effect of the isomerase or chaperone proteins (2, 22) in the catalysis of rhSCF folding. I-4 and
I-5, which are disulfide-linked dimers, are in equilibrium with other
intermediates including R. These off-pathway intermediates are
kinetically identifiable and play less important roles in the in
vitro folding of rhSCF. The observation that R, I-1, I-2, and I-3
exist as dimeric molecules appears to indicate that dimer formation
together with a small extent of secondary and tertiary structural
folding occur at a very earlier folding stage.