From the Institute of Biochemistry and Cell Biology,
Shanghai Institutes for Biological Sciences, Chinese Academy of
Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of
China and the § Department of Biochemistry, Case Western
Reserve University School of Medicine, Cleveland, Ohio 44016
Received for publication, January 28, 2003, and in revised form, March 3, 2003
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ABSTRACT |
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Human insulin is a double-chain peptide that is
synthesized in vivo as a single-chain human proinsulin
(HPI). We have investigated the disulfide-forming pathway of a
single-chain porcine insulin precursor (PIP). Here we further studied
the folding pathway of HPI in vitro. While the oxidized
refolding process of HPI was quenched, four obvious intermediates
(namely P1, P2, P3, and P4, respectively) with three disulfide bridges
were isolated and characterized. Contrary to the folding pathway of
PIP, no intermediates with one- or two-disulfide bonds could be
captured under different refolding conditions. CD analysis showed that
P1, P2, and P3 retained partially structural conformations, whereas P4
contained little secondary structure. Based on the
time-dependent distribution, disulfide pair analysis, and
disulfide-reshuffling process of the intermediates, we have
proposed that the folding pathway of HPI is significantly different
from that of PIP. These differences reveal that the C-peptide not only
facilitates the folding of HPI but also governs its kinetic folding
pathway of HPI. Detailed analysis of the molecular folding process
reveals that there are some similar folding mechanisms between PIP and
HPI. These similarities imply that the initiation site for the folding
of PIP/HPI may reside in the central Insulin is a protein hormone consisting of an acid A-chain
of 21 residues and a basic B-chain of 30 amino acids (1). The three-dimensional structure of insulin has been thoroughly studied by
x-ray crystallography (2, 3) and NMR spectroscopy (4-6), showed that
the insulin monomer is a compact globule structure with a hydrophobic
core. The A-chain contains two Although the functional form of human insulin is double-chain, human
insulin was synthesized in vivo as a single-chain
preproinsulin with a signal peptide at the N terminus of the B-chain
and a connecting peptide between the B- and A-chain (15, 16). Following
cleavage of an N-terminal signal sequence in the endoplasmic reticulum, the nascent polypeptide folds and is packaged into secretary granules as a proinsulin. Its C terminus of B-chain connects the N terminus of A-chain by the connecting peptide consisting of two bibasic amino
acids at each end of the C-peptide of 31 amino acids (17, 18).
The amino acid sequence of human proinsulin
(HPI)1 is shown in Fig.
1. Processed by a specific set of
protease, proinsulin is converted into insulin and C-peptide in the
B-cell granule (19, 20). Due to the flexibility of the C-peptide, there
is still no report of the crystal or solution structure of the
proinsulin, although proinsulin could be co-crystallized in a 1:1
complex with insulin (21, 22). Models of HPI structure were proposed in
the 1970s, suggesting that the flexible C-peptide runs across the
surface of A-chain (22, 23). Weiss et al. (24) also showed
by comparative 1H NMR and photochemical dynamic nuclear
polarization studies that the insulin moiety of proinsulin is similar
to insulin and that the connecting peptide is largely unstructured.
-helix of the B-chain. The
formation of disulfide A20-B19 may guide the transfer of the folding
information from the B-chain template to the unstructured A-chain.
Furthermore, the implications of this in vitro refolding
study on the in vivo folding process of HPI have been discussed.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helices
(IleA2-ThrA8 and
LeuA13-TyrA19, designated helix 2 and helix 3, respectively) and the B-chain consists of a central
-helix
(SerB9-CysB19, designated helix 1), flanking
turns, and extended N- and C-terminal segments. In the native
structure, the N terminus of the A-chain is closely proximate to the C
terminus of the B-chain. A and B chain are tethered by two interchain
disulfide bridges (A7-B7 and A20-B19). The third disulfide bridge
(A6-A11) is intra-A-chain disulfide. The three disulfide bonds are
important in maintaining the native conformation and biological
activities of the insulin molecular. The contributions of these bridges
to the structure, stability, and activity of hormone have been
investigated in analogues lacking selected disulfide bridges
(7-12). Internal cystine A6-A11 is close to the hydrophobic
core and its substitution with Ser or Ala resulted in the unfolding of
helix 2 (10, 11). The interchain disulfide linkage A7-B7 is fully
exposed on the surface, and its substitution with Ser leads to a marked
decrease in the thermodynamic stability and extent of folding as
compared with that of the substitution of internal cystine
A6-A11 (7, 13). NMR studies showed that all of helix 2 and part of
helix 3 were unstructured in
[SerA7,SerB7]DKP insulin and therefore
resulted in a looser conformation of the overall molecular (7). Whereas
removal of A7-B7 or A6-A11 results in only segmental unfolding,
cystine A20-B19 appears to be integral to the overall structure
and necessary for the biosynthetic expression (13). Disulfide isomers
retaining this core disulfide bridge exhibit native-like partial folds
with nonnegligible biological activity. Such isomers provide examples
of kinetic traps in an energy landscape (7-9, 14).
View larger version (29K):
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Fig. 1.
Amino acid sequence and disulfide linkage
pattern of HPI. Amino acids are shown in one-letter code, and
cysteine residues are shown as dark solid
circles. For convenience of description, the nomenclature of
the amino acids in HPI refers to that of insulin when related to the A-
and B-chain. The arrows indicate the potential cleavage site
of endoproteinase V8. Peptide fragments obtained after enzyme
cleavage are designated as F1-F9, assuming that the disulfide
bonds are reduced.
Since the discovery by Anfisen in 1960s (25) that the folding
information of proteins resides in the primary amino acid sequences,
great efforts have been devoted to the study of the molecular mechanism
of protein folding. Two important questions in the mechanism of protein
folding are still not clear: (i) how the folding is initiated and (ii)
what pathway it follows. For the latter question, one can
characterize the intermediates appearing in the folding process (26,
27). Disulfide linkages are always important factors in maintaining the
native conformation of many single-domain globule proteins
containing disulfide bonds, and their pairings are always coupled to
the formation of the functional structure during the oxidized refolding
of such proteins (28, 29). Thus, the formation of disulfide bonds could
be used as a useful tool in probing the protein-folding pathway (30).
The folding pathways of many proteins, such as bovine pancreatic
trypsin inhibitor (31-35), ribonuclease A (36-38), epidermal growth
factor (39, 40) have been widely studied by using this method. Several members of the insulin superfamily have been identified, including insulin, IGF-I (41), IGF-II (42), relaxin HI (43), amphioxus insulin-like peptide (44), prothoracicotrophic hormone-II (45), molluscan insulin-related peptide (46), Caenorhabditis
elegans insulin-like peptide (47), etc. Among these members, only
the single-chain IGF-I has been extensively investigated into its disulfide-forming pathway (48-53). Although insulin is one of the best
characterized proteins in their structure and function, its in
vitro disulfide folding pathway remains far from understood due to
the difficulties brought by its double-chain structure. Early studies
by Steiner et al. (54) have demonstrated that proinsulin
exhibits the expected property of spontaneously reforming its native
structure in alkaline solution after complete reduction in urea, which
supported the view that the single-chain proinsulin is the
biosynthetic precursor of insulin. We recently studied the
disulfide-forming pathway of a single-chain porcine insulin precursor
(PIP) and suggested that reduced PIP proceeds through two pathways to
reach the native PIP (55). Since insulin is synthesized and folded as a
single chain proinsulin in vivo (16), we further studied the
in vitro disulfide-forming pathway of human proinsulin. The
results have shown that there are some differences in the folding
behavior between HPI and PIP, indicating that C-peptide has a profound
impact on the molecular mechanism of HPI refolding. The studies on the
refolding process of the captured intermediates of HPI, on the other
hand, show that some fundamental similarities exist in both folding
mechanism of HPI and PIP. We demonstrate that the length of the
connecting peptide has a profound effect on the mechanism of folding.
Whereas PIP folds in distinct steps via one- and two-disulfide
intermediates (55), HPI is first caught in kinetic traps, forming at
least four nonnative disulfide isomers, which then rearrange to the
native state.
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EXPERIMENTAL PROCEDURES |
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Materials-- Recombinant HPI with purity greater than 98% was kindly provided by Lilly and confirmed by HPLC on a reverse phase C8 column. Protein concentration of HPI, reduced/denatured HPI, and all of the intermediates was quantified by a spectrometer and calculated with the absorption constant A278 (1 cm, 1.0 mg/ml) = 0.65 according to Ref. 56. Endoproteinase Lys-C and V8 were of sequencing grade and purchased from Sigma. The snakeskin dialysis tube with a molecular mass cut-off of 3,500 daltons was the product of Pierce. IAA, GSH, and GSSG were ultrapure and obtained from Amerresco. Ultrapure dithiothreitol and L-Arg were purchased from Sigma. Ultrapure urea and guanidine-HCl were the products of Promega. Acetonitrile and trifluoroacetic acid were of HPLC grade. All of the other reagents used in the experiment were of analytical grade.
Refolding of Fully Reduced/Denatured HPI
(frdHPI)--
Purified HPI was dissolved in 100 mM
Tris-HCl buffer (pH 8.7), containing 8 M guanidine-HCl, 1 mM EDTA, and 100 mM dithiothreitol. Reduction
of the protein was carried out at 37 °C for 30 min. Thereafter, the
buffer was immediately exchanged to 10 mM HCl by gel
filtration using Sephadex G-25 (Amersham Biosciences) and stored at
80 °C for later use. The refolding of reduced and denatured protein was initiated by diluting the frdHPI with the refolding buffer
(100 mM Tris-HCl, 5 mM EDTA, different
concentrations of GSH and GSSG, with or without L-Arg) at a
final protein concentration of 0.1 mg/ml. The refolding reaction was
carried out at 16 °C for 16 h and quenched by adding
trifluoroacetic acid to pH 1.0, and then the mixture was
analyzed on HPLC using a C4 reverse phase column to quantify the
refolding yield. The impacts of different redox constitutions, pH, and
L-Arg on the refolding yield of HPI have been tested, and
an optimized refolding condition with the best yield of HPI was
selected to carry out the in vitro studies of the
disulfide-refolding pathway of HPI.
Trapping, Separation, and Purification of Refolding Intermediates-- Refolding of frdHPI was initiated by adding frdHPI in the prewarmed (16 °C) refolding solution (100 mM Tris, pH 10.0, 5 mM EDTA, 1 mM GSH, 5 mM GSSG) at a final concentration of 0.1 mg/ml. The refolding process was quenched by acid or iodoacetic acid in a time course manner. For the acid quenching method, aliquots of protein solution were removed at different refolding times and quickly mixed with an equal volume of 2% trifluoroacetic acid to lower the pH to 1.0. For the iodoacetic acid method, the refolding reaction was quenched by adding one-fourth volume of freshly prepared 0.5 M IAA in 50 mM Tris-HCl (pH 8.5), and the carboxymethylation reaction was performed at room temperature for 5 min. To observe the temporal distribution of refolding intermediates, the IAA-quenched solution was adjusted to pH 1.0 with trifluoroacetic acid and immediately analyzed by HPLC. For large scale preparation of the trapped intermediates, the solution containing IAA-trapped intermediates was dialyzed against 50 mM NH4HCO3 (pH 8.0) at 4 °C and thereafter lyophilized. Then the refolding mixture was separated by HPLC on a reverse phase C4 (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) column. Unless otherwise indicated, the solvent A used in HPLC was water containing 0.15% trifluoroacetic acid, and the solvent B was 60% acetonitrile containing 0.125% trifluoroacetic acid. The linear elution gradient was 40-80% of solvent B in 30 min with a flow rate of 0.5 ml/ml, and the monitoring wavelength was 280 nm. The corresponding peaks were collected manually and lyophilized. Then the partially separated intermediates were further purified by a semiprepared C8 (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) column and characterized by an analytical C8 column (ZORBAX SB, 4.6/150 mm; DuPont) on reverse-phase HPLC.
Disulfide Linkage Analysis of the Intermediates by Enzyme
Digestion--
Two endoproteinases, Lys-C and V8, were utilized to
digest the intermediates of HPI in order to characterize their
disulfide linkages. Lys-C and V8 cleave at the carboxyl terminus of Lys and Glu, respectively. There are two lysine residues and eight glutamic
acids in the amino acid sequence of HPI. In general for the enzyme
digestion, 10 µg of the intermediates was dissolved in 10 µl 100 mM NH4HCO3 (pH 8.0), and 1.5 × 103 units of Lys-C or 0.5 µg of V8 was added. HPI
was used as a positive control in each of the enzyme digestion. The
reaction was carried out at 25 °C for 16 h and quenched by
adding 90 µl of 0.3% trifluoroacetic acid. Then the digestion
mixture was immediately analyzed by reverse-phase HPLC on C8 column
(ZORBAX SB-C8, 5µ, 4.6/150 mm; DuPont). The elution gradient was
25-65% of solvent B linear in 35 min. The flow rate was 0.5 ml/min,
and the detector wavelength was set at 210 nm.
Folding of the Intermediates-- Isolated intermediates of HPI with three isomeric disulfide bridges were dissolved in 5 mM HCl, and the protein concentration was adjusted to 0.2 mg/ml. Before the folding of the intermediates, protein solution and a 2-fold concentration of refolding buffer (200 mM Tris, pH 10.0, 10 mM EDTA, 2 mM GSH, 10 mM GSSG) were incubated at 4 °C for 10 min, respectively. The folding reaction was initiated by mixing them with equal volume and carried out at 4 °C. Aliquots of the folding solution were removed in a time course manner and mixed with an equal volume of 2% trifluoroacetic acid to stop the folding process. Then the mixture was analyzed by reverse-phase HPLC on a C4 column (Sephasil peptide, ST 4.6/250 mm; Amersham Biosciences) with a linear gradient of 50-80% of solvent B in 30 min. The flow rate was 0.5 ml/min, and the detector wavelength was 230 nm.
Circular Dichroism Studies--
All of the samples were
dissolved in 10 mM HCl except for the IAA-modified frdHPI
in 50 mM NH4HCO3 (pH 8.0). The
protein concentration was all adjusted to 0.25 mg/ml by UV absorption
at 278 nm. Circular dichroism measurements were performed on a
Jasco-700 circular dichroism spectropolarimeter at 25 °C. For the
far-UV CD spectra, samples were scanned from 190 to 250 nm and
accumulated twice at a resolution of 1.0 nm with a scanning speed of 50 nm/min. The cell length was 0.1 cm, and the stepwise increase
was 0.1 nm. For the near-UV CD spectra, samples were scanned from 250 to 310 nm and accumulated twice at a resolution of 1.0 nm with a speed
of 50 nm/min. The cell length was 1.0 cm. All of the CD data were
expressed as mean residue ellipticity. The secondary structure contents
of the samples were estimated by using the software J-700 for Windows
Secondary Structure Estimation, Version 1.10.00 (JASCO Corp.), equipped
with the Jasco-700.
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RESULTS |
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In Vitro Refolding of HPI with High Yield-- To optimize the refolding conditions, the concentration of GSH and GSSG, the pH, and the absence or presence of the L-Arg have been tested. After a series of tests, we have found that several factors in the optimized refolding condition of HPI are obviously different from those of PIP, although the difference of two molecules in sequence is only the length of connecting peptide. First, L-Arg was not absolutely required for the refolding of HPI, whereas the yield of PIP was lower than 15% in the absence of L-Arg. Even oxidized by air, HPI could fold spontaneously with a high yield up to 50%. Thus, it is clear that HPI refolds more efficiently than PIP in vitro. Second, predominance of GSSG in the redox buffer is required for the high yield of HPI, whereas GSH is required for PIP. Table I shows the recovery of refolded native HPI reached as high as 84% when the frdHPI was diluted in the refolding buffer containing 100 mM Tris, pH 10.0, 5 mM EDTA, 1 mM GSH, and 5 mM GSSG at a final concentration of 0.1 mg/ml and incubated at 16 °C for 16 h. Thus, the optimum was used in the in vitro refolding study of HPI.
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Temporal Distribution of the Disulfide Intermediates of
HPI--
After the refolding process was initiated by adding frdHPI
into the refolding buffer, the reaction was quenched at different time
points by lowering the pH to 1.0 with trifluoroacetic acid, and
the mixture was analyzed immediately by reverse-phase HPLC on a C4
column. The temporal distribution of the refolding intermediate species
is shown in Fig. 2. Four obvious
intermediates, designated by elution time, P1, P2, P3, and P4, were
observed during the refolding process of HPI. In the first 2 min
of the refolding, only a very small proportion of the intermediates as
well as the frdHPI can be identified by HPLC. The SDS-PAGE results
(data not shown) of the deposits and the supernatants of the
acid-quenched refolding mixture showed that intermediates during this
period were easily acid-precipitated. Analysis of the precipitate of the refolding mixture at different time points by SDS-PAGE (data not
shown) showed that they were mostly disulfide-linked intermolecular aggregation. These aggregated species always act as off-pathway intermediates and hence prevent the HPI refolding efficiency to reach a
full scale. After 2 min of the refolding process, HPI and the four
intermediates begin to dominate the folding intermediate species. Fig.
3A shows the recovery curve of
the native HPI calculated from the HPLC chromatography, and Fig.
3B shows the time-dependent distribution of the
four intermediates. The contents of all of the intermediates came to a
climax at 6 min, and then the content of P1 decreased slowly, whereas
P2 and P4 decreased more quickly. Although there are some minor
intermediates other than P1, P2, P3, and P4 at 2 min of the
folding, it is very difficult to identify them due to the lower
contents.
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The refolding process was also quenched by IAA and observed by HPLC as shown in the right lane of Fig. 2. In contrast with the acid-quenched results, there are only three major intermediates peaks, designated as IA1, IA2, and IA3, respectively, along the refolding. The elution time of IA1 and IA2 is the same as that of acid-trapped P1 and P2. IA3 was later confirmed by native electrophoresis to be a mixture of acid-trapped P3 and P4. The lower resolution of HPLC in IAA-trapping than acid-trapping was probably caused by IAA.
Populated Intermediates Are Disulfide Isomers--
The refolding
process was quenched by trifluoroacetic acid at 6 min, and the mixture
was separated by HPLC on a C4 column. The partially separated
intermediates, P1, P2, P3, and P4, were modified by IAA and purified by
HPLC on a C8 column. Then the molecular mass of the purified
IAA-modified intermediates was measured by ESI-MS, and all of them were
shown to be 9388, which was identical to that of native HPI. This
indicates that all of the intermediates have no reaction with IAA and
that P1, P2, P3, and P4 contain three intact disulfide
bonds, with at least two nonnative ones. Therefore, the four
intermediates are scrambled disulfide isomers of HPI. These
purified intermediates were further analyzed by pH 8.3 native
electrophoresis, as shown in Fig. 4. P1,
P2, and P3 were a little slower than native HPI in the electrophoresis, indicating that they have a looser conformation than HPI. P4 was much
slower than native HPI and the other intermediates, so it has a much
more flexible conformation. Although the purified intermediates were
symmetrical peaks on HPLC profiles as shown later, there are some bands
at the lower portion of each lane of the intermediates on pH 8.3 native
electrophoresis. These minor bands were probably the results of
deamidation of the intermediates, which was observed in our
previous result (55).
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For large scale preparation of the four intermediates, the refolding process was stopped by adding IAA that will modify all of the free thiols. Then the mixture was dialyzed, lyophilized, desalted, and separated by RP-HPLC on a C4 column. Because the four intermediates are close in the elution profile, it is very difficult to thoroughly separate them by HPLC once. So, we further purified them with two rounds of RP-HPLC on a semiprepared C8 column. The purity and elution time were identified by RP-HPLC on an analytical C8 column.
Disulfide-linkage Elucidation of the Four Intermediates--
Four
intermediates were first digested by endoproteinase Lys-C, which
cleaves at the carboxyl terminus of lysine residues (B29 and C34), and
analyzed by RP-HPLC on a C8 column as shown in Fig.
5. All of the major peaks of the mixture
of the enzyme-digested intermediates on HPLC were collected, and their
molecular weight was measured by ESI-MS as shown in Table
II. The left peaks of each profile in
Fig. 5 (HPI-Fa, P1-Fa, P2-Fa, P3-Fa, and P4-Fa) correspond to the
fragment containing the C-peptide. The molecular weights of another two
peaks in Lys-C-digested P4, peaks P4-Fb and P4-Fc, correspond to the
fragments of A- and B-chain with intrachain disulfide bridges,
respectively. This indicates that P4 should have a nonnative B9-B19
disulfide bond and no inter-AB disulfide linkages. Combined with the
results from Fig. 4, P4 is the most flexible isomer with a B7-B19
intra-B-chain bridge. Lys-C-digested P1-P3 in Fig. 5 showed the same
molecular weight of 5862 for peaks P1-Fb, P2-Fb, and P3-Fb,
implying that P1-P3 are three disulfide isomers with two interchain
disulfide bridges.
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To further elucidate the disulfide linkage of the four intermediates,
endoproteinase V8 that cleaves at the carboxyl terminus of Glu residues
was utilized to digest these intermediates. Because native HPI contains
eight Glu residues and two inter-AB disulfide bonds (A7-B7 and A20-B19)
each link two pieces of fragments as indicated in Fig. 1, only seven
fragments would be obtained after V8 digestion. After the enzyme
digestion, the mixture was separated by HPLC, and each profile is shown
in Fig. 6. The major peaks were
collected, and their molecular weight was measured by ESI-MS as shown
in Table III. From mass values shown in
Table III, the corresponding fragment of each peak on HPLC could be
identified. Peaks a', a1', a2', a3', and a4' in Fig. 6
correspond to fragment F5 in Fig. 1. In peak a, a1-a4 correspond to
F7. In peak b, b1-b4 belong to F3, and in peak d, d1-d4 are F6. All
of these four peaks (a', a, b, and d) correspond to the segments
without disulfide bonds. V8-digested P1 showed these four peaks and
other small peaks, which are similar to c2, e2, f3, and f4 in
three bottom profiles in Fig. 6. This
suggests that P1 may be a mixture of several intermediates with
interchain disulfide bonds. The molecular weights of peaks c2 and e2 of
V8-digested P2 are identical to those of peak c and e of V8-digested
HPI, respectively, but the retention time of peak e2 is shorter than
that of peak e of HPI. This demonstrates that P2 contains the disulfide
bond A20-B19 and another nonnative inter-AB one, possible B7-A6 or
B7-A11. For the V8-digested P3, the major peak f3 with a molecular
weight of 1993 corresponds to that of fragment F1 linked with F9
of HPI. Accordingly, it could be expected that P3 contains a nonnative B7-A20 disulfide linkage. For the V8-digested P4, in addition to peak
a4, b4, and d4 as described above, peak f4 has a molecular weight of
2347 that was the sum of the molecular weight of fragments F1 and F2 in
Fig. 1. This indicates that P4 contains the intra-B-chain disulfide
B7-B19. Due to the absence of amino acid sequencing of the
intermediates, we could not distinguish the disulfide linkage patterns
among A6, A7, and A11. All of the possible disulfide linkages of
intermediates P2-P4 are shown in Fig. 7.
For intermediates P3 and P4, they may be one of these three possible
disulfide linkages or the mixture of them. The disulfide linkage
pattern of P2 has two possibilities, among which isomer a and b
correspond to the insulin isomer swap and swap-2,
respectively (57, 67). There are a total of 14 possibilities of the
disulfide isomers of HPI, and P1 may be a mixture of all or part of the
other isomers except for P2, P3, and P4.
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Intermediates are Partially Folded--
The far-UV CD spectra of
the four intermediates are shown in Fig.
8A. The results show that
there is obvious reduction in -helix content of all these
intermediates relative to native HPI. Calculated from the CD spectra of
each sample, P1-, P2-, P3-, P4-, frdHPI-, and IAA-modified frdHPI
retained approximately helical contents of 40, 40, 55, 12, 22, and 0%
related to total helices of native HPI, respectively. Because HPI
contains 27 helical residues, the number of helical residues in P1-P4
is in turn estimated to be 11, 11, 15, and 3, respectively.
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The near-UV CD spectra of HPI, P1, P3, P4, and frdHPI are shown in Fig. 8B. Intermediate P2 has not been measured, because we are unable to get enough samples for this analysis. From their spectra, P4 is most similar to frdHPI, whereas P1 and P3 are much closer to HPI than P4. This indicates that P4 has the less folded conformation, whereas P1 and P3 with the interchain disulfide bonds have the partially structured conformation.
Reassortment of the Disulfide Isomeric Intermediates--
All of
the purified intermediates with the three isomeric disulfide bridges
were reconstituted into the refolding buffer to allow the reshuffling
of the disulfide bonds to native forms. Refolding was quenched by
trifluoroacetic acid and immediately analyzed on HPLC in successive
times in Fig. 9, in which all four intermediates could refold into native HPI with the correct disulfide pairing. This indicates that P1, P2, P3, and P4 are on-pathway intermediates along the folding process of HPI. P2 with A20-B19 native
interchain bridge provides the most routes to the native state. There
are three lines of evidence. (i) The refolding speed of P2 is faster
than the other three intermediates. After 15 min of refolding, more
than 50% of the P2 has been converted into the native HPI. (ii) The
refolding process of any intermediates is always concomitant with the
appearance of the other three intermediates. However, during the
refolding of P2, the proportion of the other intermediates is the
lowest among all of the intermediates. (iii) P2 accumulates during the
refolding process of P1, P3, and especially P4. Although P1 is closer
to native HPI than P2 at elution time on HPLC, P2 is still a major
intermediate during the disulfide rearrangement of P1. These
observations suggest that P2 with one native disulfide bridge,
A20-B19, is the most direct intermediates to reach the native HPI. P1,
P3, and P4 may have to rearrange to form P2 before reaching the native
HPI.
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DISCUSSION |
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Disulfide Folding Pathway of HPI and the Possible Molecular Mechanism-- During the refolding process of HPI, only scrambled disulfide isomers with three disulfide bonds were captured as intermediates. Unlike the refolding of PIP, there are no accumulated intermediates with one or two disulfide bridges identified. It is possible that the different refolding behavior between HPI and PIP might have been caused by the different conditions, such as the excess GSSG over GSH in the redox buffer in HPI refolding we used. To check this possibility, we carried out the refolding process of HPI under the similar situation with that of PIP, in which the refolding buffer contained 50 mM Tris, pH 9.5, 0.5 M L-Arg, 5 mM EDTA, 5 mM GSH, 0.5 mM GSSG, and the protein concentration was 0.1 mg/ml. Time-dependent distribution of the intermediates was observed on HPLC, and there were still no additional detectable intermediates (data not shown). This implies that the different patterns of intermediates accumulated along the refolding process of HPI and PIP are not caused by different refolding conditions used but are determined by the inherent properties of the distinct proteins.
Of the four intermediates of HPI, those with two interchain disulfide bonds, such as P1, P2, and P3, always possess partially structured conformation, as deduced from the results of native gel electrophoresis and CD spectra. Only P4 without inter-AB disulfide bridges has a very flexible conformation, which is similar to the frdHPI. This indicates that the formation of interchain disulfide linkages, whether native or nonnative, is a crucial factor to construct the native conformation.
For the single-domain protein with disulfide bridges, the hydrophobic interaction and the covalent disulfide linkage are two important forces to maintain the native conformation and direct the folding pathway of a protein (30). The oxidative refolding pathway of HPI is also governed by the mutual interaction of the two forces. The fact that the accumulated scrambled disulfide isomers are refolding intermediates and an equilibrium among the intermediates of HPI suggests that the folding of HPI probably adopts the following mechanism. At the very beginning of the HPI refolding, where the hydrophobic interaction has not contributed to the folding of the peptide chain, the nearest free thiols on the random-coiled frdHPI paired randomly and rapidly to form the three scrambled disulfide linkages, and most of them are nonnative as the intermediates of P4. Subsequently, driven by the hydrophobic interaction, the secondary structure is gradually formed in the intermediates, which initiate and direct the reshuffling of the nonnative disulfide bonds to form the native disulfide bonds and consequently solidify the partially formed conformation. Accordingly, the intermediates, such as P1, P2, P3, and P4, with nonnative disulfide bridges were captured in the early stage of the HPI refolding.
Refolding of the four intermediates suggests that P2 possessing native
disulfide bond A20-B19 is the most important intermediate during the
disulfide reshuffling process of HPI, and the other three intermediates
must rearrange their disulfide pairings to form P2 on their way to the
native HPI. Based on the observations and deductions above, we have
proposed a putative disulfide folding pathway of HPI in
vitro as shown in Fig. 10. In the
absence of oxidized reagent such as GSSG, free thiols of frdHPI can
also pair randomly and rapidly to form in turn the one-disulfide and two-disulfide intermediates until consequently native HPI or the three-disulfide scrambled isomers such as P1, P2, P3, and P4. Thereafter, the disulfide bonds in the isomers of intermediates begin
to reshuffle. P4 without the interchain disulfide linkage could convert
directly, or indirectly by way of P1 or P3, into the P2 by disulfide
rearrangement. P1 and P3 could interconvert each other, and both of
them could convert into P2 before they finally refold into the native
HPI.
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NMR structure analysis of the insulin analogues lacking the disulfide bridge A7-B7 or A6-A11 showed that B-chain retains native-like supersecondary structure, whereas most of the A-chain is disordered (7, 10, 12). These observations suggest that the B subdomain provides a template to guide folding of the A-chain. Accordingly, we suggest that the disulfide reshuffling during the folding of HPI was also a process directed by the folding information of B-chain templates. The partially structured P1, P2, and P3 should have a native-like B subdomain with intact helix 1. The folding information in the preformed B-chain was transferred to the A-chain by means of C-peptide and interchain disulfide bridges, and hence the folding of the A-chain guides the disulfide reshuffling in these intermediates. For the P4 with a very felxible conformation, the intra-B chain disulfide bridge B7-B19 may be a hindrance to form the central helix B9-B19. Only after the untethering of the disulfide B7-B19, the native-like structure in the B-chain can be formed and consequently guide the folding of A-chain as a template and reshuffling of nonnative disulfide linkage.
Formation of the A20-B19 Bridge Is the Most Important Step in the Folding Pathways of Both PIP and HPI-- Some differences exist between PIP and HPI in their disulfide refolding pathways. First, only three intermediates with one or two disulfide bonds but not with three disulfide bonds were captured during the oxidized refolding of PIP (55). However, four intermediates with three disulfide bonds were identified during the oxidized refolding process of HPI. Second, most of the disulfide bonds in the PIP intermediates are native, whereas those in HPI are nonnative. Third, the rate-limiting step during the refolding of PIP resides in the conversion of the 2SS intermediate into the native PIP, whereas for HPI, the rate-limiting step was the disulfide reshuffling in the 3SS isomers.
Nevertheless, intrinsic similarities of refolding mechanism also exist between PIP and HPI. We have proposed two disulfide forming pathway of PIP during its refolding in vitro. One begins with the first formation of A6-A11, and the other begins with A20-B19 (55). Contrary to the first formation of A20-B19 requiring the long range interaction in the reduced polypeptide, disulfide linkage A6-A11 is the first favored possibly for the proximity of these cysteines in the sequence, and hence this pathway will eventually shift to the pathway in which A20-B19 first forms. Two partially structured intermediates, 2SSPIPa and 2SSPIPb, both have the disulfide bond A20-B19. Therefore, formation of A20-B19 seems to be a crucial step in the folding of PIP, because the A20-B19 disulfide bond participates in and stabilizes a molten subdomain, which is important for the initiation folding of PIP. During the refolding of HPI, P2 is the most important one among the four captured intermediates because of its faster speed and lower proportion of other intermediates during its rearrangement to HPI. All of the other three intermediates may have to reshuffle their disulfide bonds to form P2 before they can reach native HPI. Given that P2 contains a native A20-B19 disulfide bond, it is reasonable to conclude that formation of A20-B19 is an important factor along the folding pathway of HPI. The possible role of the formation of A20-B19 in the disulfide isomers is to anchor the A- and B-chain of HPI in the native-like orientation. Therefore, in the both refolding pathway of PIP and HPI, the formation of A20-B19 is a crucial step.
The pairwise substitution of internal disulfide A20-B19 with serine in PIP resulted in expression of a product that was too low to be detectable (13). This further suggests that A20-B19 bond is important in the folding pathway and maintaining its native conformation. Recently, a PIP mutant retaining only one disulfide, A20-B19, has been successfully expressed in yeast (71). CD results showed that it retained certain secondary structure, and the in vitro refolding experiment showed that CysA20 and CysB19 could pair rapidly with high yield. This peptide model of folding intermediates clearly demonstrated that A20-B19 is the most important one among the three disulfide bonds.
Folding Initiation Site of HPI/PIP-- One of the most important questions in protein folding is how the folding reaction is initiated (57). The folding rates and mechanisms appear to be largely determined by the topology of the native/folded state, whereas the initiation of protein folding always requires several residues that are located in the sequence to form a nucleation site as an initiation site of folding (58, 59). Although the structure and function of insulin have been amply documented, the folding initiation site of insulin/HPI/PIP is rarely studied.
The disulfide linkage patterns and native -helix content of the
partially structured intermediates of PIP and HPI are listed in Table
IV, and the peptide models of the insulin
folding intermediates whose structures have been studied by NMR are
listed in Table V (7, 9, 10, 12). By
comparing the peptide models with our captured intermediates here, we
could infer that all of the partially structured intermediates of PIP
and HPI retain helix 1. Therefore, it is reasonable to suggest that
helix 1 is firstly formed during the folding of PIP and HPI. The
formation of helix 1 in the B-chain contributes substantially to the
native-like conformation of the B-chain that will guide the folding of
A-chain. Thus, the folding initiation site of PIP and HPI should reside in the central helix 1 in which some conservative hydrophobic residues,
such as LeuB11, ValB14, and LeuB15,
could be the major components of the initiation site. The hydrophobic collapse of these residues at an early stage of folding forms the
initiation site of HPI/PIP.
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Potential Role of the C-peptide in the Refolding of HPI-- As shown under "Results," two major differences exist between the folding behavior of PIP and HPI. First, the in vitro refolding yield of HPI could easily come to a high level, whereas it is difficult for PIP to refold efficiently in the same condition. Second, HPI and PIP adopt different disulfide-forming pathways during the in vitro folding. Because the only difference of sequence between PIP and HPI is that the connecting peptide in which PIP is a dipeptide (Lys-Ala), whereas that of HPI is a C-peptide plus cleavage site at each end, so it is the connecting peptide (mainly the C-peptide) of HPI that was responsible for the discrepancy in folding behavior with PIP.
How the flexible C-peptide contributes to the high refolding yield of HPI? The 31-residue C-peptide contains five acidic amino acids and no basic ones, so it is a negative charge-rich peptide fragment. The concentrated charge in the peptide might result in the intermolecular repulsion during the refolding of HPI and hence attenuated the intermolecular hydrophobic interaction that always leads to the aggregation. Therefore, the acidic residues in C-peptide may account for the higher refolding yield of HPI. Our results are consistent with a recent experiment in which both alanine replacement mutation and deletion of three highly conserved acidic residues (EAED) at the N terminus of the C-peptide resulted in serious aggregation during HPI refolding (60). Therefore, it is possible that the C-peptide acts as an intramolecular chaperone in the folding of HPI. Actually, there are many examples of the pro region of the protein precursor facilitating the folding of the precursor as an intramolecular chaperone, of which pre-bovine pancreatic trypsin inhibitor is a well characterized one (61). Another role of the flexible C-peptide is probably related to allowing sufficient flexibility in the positioning of the chains for their optimal interaction. Thus, the constraints imposed by the short C-peptide in PIP, especially on the N-terminal region of the A chain, may disrupt the correct intra- and interchain interactions that sort out this region after the A20-B19 disulfide bridge forms. The fact that IGF-I tends to have folding isomers (52) lends support to the view that a shortened C-peptide constrains the folding process within the N-terminal A domain. There, the C-peptide of HPI seems to also act as a flexible tether and thus enhance folding by facilitating the molecular interaction between the A- and B-chain of HPI.
Although the C-peptide has no effects on the thermodynamic folding of PIP, it has profound effects on the kinetic folding pathway. How does the C-peptide affect the folding pathway of HPI? The proline isomerization is always a rate-limiting step in the folding reaction of small, single-domain proteins containing proline residue(s) (62). PIP has one proline residue at position B28, whereas HPI has three proline residues (the other two in the C-peptide), so the conformation formation of PIP is probably faster than HPI. During the refolding of PIP, the formation of native-like conformation may be faster than HPI. The native-like conformation will consequently guide the correct and hierarchical pairing of disulfide bonds. Thus, the different folding mechanism of HPI and PIP may be partially due to the proline isomerization.
Therefore, although the A- and B-chain of insulin contain sufficient information for the correct pairing of disulfide bonds (63, 64), we consider that the intact folding information of HPI resides in the A- and B-chain of insulin as well as the C-peptide.
Implications for the in Vivo Folding of HPI-- Only scrambled isomers in the refolding of HPI have been captured as intermediates, indicating that the free thiols on frdHPI prefer to form nonnative disulfide bonds rather than stand alone during the refolding process of HPI. The thiol-free scrambled disulfide isomer could preclude the intermolecular disulfide linkages for the off-pathway intermediates. In addition, the reshuffling of disulfide bonds in the disulfide isomers is an intramolecular reaction with high efficiency. Therefore, it is reasonable that the scrambled disulfide isomers could serve as folding intermediates in many other proteins such as hirudin, TAP, PCI, and epidermal growth factor (39, 65-67).
The foldability of proinsulin is limited in vitro in the
absence of chaperones by aberrant aggregation of partially folded intermediates. Although the present studies were conducted at pH 10.0 to avoid such aggregation, the results have implications for folding
under physiological conditions. HPI is synthesized in vivo
as a preproinsulin with a signal peptide (16). The signal peptide then
targets the pre-HPI for translocation into the endoplasmic reticulum,
where the signal peptide region was cut off. Considering that
glutathione is the principal redox buffer in endoplasmic reticulum (68)
(similar to the redox condition we used here) and our results, we might
imagine the refolding of HPI in vivo may adopt the following
mechanism. During translation of the nascent peptide of HPI from
ribosome into the endoplasmic reticulum, the six cysteines will be
exposed to the endoplasmic reticulum one after another, and hence the
CysB7 and CysB19 of HPI will be first exposed.
Since the successive exposure of cysteines in A-chain must wait for the
translocation of a 35-amino acid connecting peptide, the free
CysB7 and CysB19 have the tendency to pair and
form the disulfide bond B7-B19. The consecutively exposed cysteines on
the A-chain will pair each other to form three consecutive disulfides
like the intermediate P4. Thereafter, a series of scrambled disulfide
isomers were formed like intermediate P1, P2, and P3 by disulfide bonds
reshuffling. Along with forming secondary structure, the
scrambled disulfide isomers, through reshuffling into native disulfide
bonds, finally complete the folding. Despite the higher efficiency of
HPI under the optimized conditions in in vitro experiments,
the overall folding rate of HPI in these studies is much slower
compared with the rate of protein synthesis and folding in
vivo (69). This suggests that the folding of HPI in
vivo requires the involvement of some cellular factors like
protein-disulfide isomerase and chaperone. A recent study has shown
that the catalytic activity and chaperone function of human
protein-disulfide isomerase are required for the efficient refolding of
proinsulin (70).
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ACKNOWLEDGEMENTS |
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We thank two anonymous reviewers for helpful advice and suggestions and Emily K. Collins for assistance with figures.
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FOOTNOTES |
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* This work was supported by National Foundation of Natural Science Grant 39670179 and Chinese Academy of Sciences Grant KJ951-B1-606.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 86-21-54921133; Fax: 86-21-64338357; E-mail: fengym@sunm.shcnc.ac.cn.
Published, JBC Papers in Press, March 6, 2003, DOI 10.1074/jbc.M300906200
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ABBREVIATIONS |
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The abbreviations used are: HPI, human proinsulin; PIP, porcine insulin precursor; IGF, insulin-like growth factor; IAA, sodium salt of idoacetic acid; frdHPI, fully reduced/denatured HPI; HPLC, high performance liquid chromatography; RP-HPLC, reverse phase HPLC; SS, disulfide bond; ESI-MS, electrospray ionization-mass spectrometry; HI, human insulin; DKP insulin, [AspB10, LysB28, ProB29] insulin mutant.
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