Unfolding of human proinsulin
Intermediates and possible role of its C-peptide in folding/unfolding
Cheng-Yin Min, Zhi-Song Qiao and You-Min Feng
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
We have investigated the in vitro refolding process of human
proinsulin (HPI) and an artificial mini-C derivative of HPI
(porcine insulin precursor, PIP), and found that they have
significantly different disulfide-formation pathways. HPI
and PIP differ in their amino acid sequences due to the
presence of the C-peptide linker found in HPI, therefore
suggesting that the C-peptide linker may be responsible for
the observed difference in folding behaviour. However, the
manner in which the C-peptide contributes to this difference
is still unknown. We have used both the disulfide scrambling
method and a redox-equilibrium assay to assess the stability
of the disulfide bridges. The results show that disulfide
reshuffling is easier to induce in HPI than in PIP by the
addition of thiol reagent. Thus, the C-peptide may affect the
unique folding pathway of HPI by allowing the disulfide
bonds of HPI to be easily accessible. The detailed processes
of HPI unfolding by reduction of its disulfide bonds and by
disulfide scrambling methods were also investigated. In the
reductive unfolding process no accumulation of intermedi-
ates was detected. In the process of unfolding by disulfide
scrambling, HPI gradually rearranged its disulfide bonds to
form three major isomers G1, G2 and G3. The most abun-
dant isomer, G1, contains the B7-B19 disulfide bridge. Based
on far-UV CD spectra, native gel analysis and cleavage
by endoproteinase V8, the G1 isomer has been shown to
resemble the intermediate P4 found in the refolding process
of HPI. Finally, the major isomer G1 is allowed to refold to

of denaturants [8–10]. The unfolding pathways of most
proteins have been studied using denaturation, in which the
disulfide bonds remain intact. Due to the cooperative and
interdependent role of the disulfide bonds in maintaining
the native conformation of the majority of proteins, the
reductive unfolding pathway always results in an Ôall-
or-noneÕ mechanism. Therefore, it is very difficult to capture
the disulfide intermediates and to complete additional
investigation of the molecular mechanism of the unfolding
pathways. Currently, the unfolding pathway of a limited
number proteins, such as bovine pancreatic trypsin inhi-
bitor, RNaseA and a-lactoalbumin, have been well charac-
terized by using the reductive unfolding method [11–14].
The recently established disulfide scrambling method of
Chang et al. may make it possible to dissect experimentally
the reductive unfolding of a disulfide-containing protein
into two distinct stages [15]. During the first stage, in
the presence of denaturant and trace thiol catalyst, native
Correspondence to Y M. Feng, Shanghai Institute of Biochemistry,
Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031,
China. Fax: + 86 021 54921011, Tel.: + 86 021 54921133,
E-mail:
Abbreviations: HPI, human proinsulin; PIP, porcine insulin precursor;
IGF-I, insulin-like growth factor-I; TAP, tick anticoagulant peptide;
PCI, potato carboxypeptidase inhibitor; LCI, leech carboxypeptidase
inhibitor; GdnHCl, guanidine hydrochloride; IAA, sodium salt of
idoacetic acid; GSH, reduced glutathione; GSSG, oxidized gluta-
thione; frdHPI, fully reduced/dentured HPI; frHPI, fully reduced HPI;
ESI-MS, electrospray ionization-mass spectrometry.
Note: C Y. Min and Z S. Qiao contributed equally to this work.

bonds intact [27,28]. By using near- and far-UV CD, Brems
et al. have investigated the guanidine hydrochloride-
induced equilibrium denaturation of insulin and proinsulin
[29,30]. The results of previous work on insulin are
consistent with a two-state denaturation process that lack
any appreciable equilibrium intermediates. The character-
ization of the unfolding of insulin and proinsulin using the
reductive unfolding method has not been thoroughly
investigated. We have characterized the unfolding process
of an artificial porcine insulin precursor (PIP), in which a
dipeptide, AK, links the B and A chain, as shown in
Fig. 1A, in denaturants containing a thiol catalyst. We
observed that PIP reshuffled its native disulfide bonds to
form disulfide isomers, with one major disulfide isomer
present. The disulfide isomers of PIP could spontaneously
refold to native PIP in the presence of a thiol reagent, clearly
demonstrating that PIP has only one thermodynamically
stable form [31]. Recently, the in vitro refolding process of
human proinsulin (HPI) has been investigated in our
laboratory. Four scrambled disulfide isomers with three
intact disulfide bonds have been captured as intermediates
[32]. To compare the disulfide isomers that appeared during
the refolding and unfolding of HPI, we have investigated the
process of unfolding by using disulfide scrambling method
as well as the denaturation method. These results show a
striking correlation between the oxidized refolding and
unfolding of HPI by the disulfide scrambling method.
HPIisthenativein vivo precursor of human insulin in
which the B-chain and A-chain are connected by a flexible
31 residues connecting peptide (C-peptide), as shown in

difference between PIP and HPI in the refolding pathway.
However, our studies of the oxidized refolding process of
PIP and HPI in vitro [32,35] have found that these two
proteins adopt two significantly different disulfide forming
pathways as shown in Fig. 1B (PIP) and 1C (HPI). As a
result, we can conclude that the connecting peptide in HPI
partially controls its unique folding behaviour. However,
the manner in which flexible C-peptide contributes to this
folding process is still unknown. Compared with the step-
by-step formation of the disulfide bonds in PIP (Fig. 1B),
disulfide bond formation in HPI occurs by random
formataion of intramolecular disulfide bonds at the begin-
ning of oxidized refolding, and then rearrangement from
non-native to native disulfide bonds. This different folding
behaviour indicates that the energy state of the disulfide
bonds in HPI and PIP may not be similar. During the HPI
unfolding studies here, the disulfide scrambling method and
redox equilibrium assays were used to test this hypothesis.
The results confirm that the disulfide bond stability of
HPI is lower than that of PIP, which indicates that the
C-peptide may control the folding behaviour of HPI by
making the disulfide bonds more accessible.
Experimental procedures
Materials
Recombinant HPI and PIP were of > 98% purity as
confirmed by RP-HPLC on a C8 column. Endoproteinase
Lys-C and V8 were of sequencing grade (Sigma). The
sodium salt of iodoacetic acid (IAA), reduced glutathione
(GSH) and oxidized glutathione (GSSG) were ultra pure
(Amersham Biosciences, Piscataway, NJ). Ultra pure

threitol in the above buffer for 16 h at 25 °C. To confirm the
identity of the reduced protein, frHPI was modified by IAA
and then separated by native PAGE. The native PAGE
showed that there was only one single band, suggesting that
disulfide bonds in HPI were fully reduced. The fully
reduced/denatured HPI (frdHPI) was obtained by reducing
the native HPI with dithiothreitol in the presence of 6.0
M
guanidine hydrochloride (GdnHCl), as described in our
previous work [32].
Unfolding of HPI in the presence of denaturant
and thiol catalyst
The native HPI was dissolved in buffer containing 100 m
M
Tris pH 8.7, 1 m
M
EDTA, 0.2 m
M
2-mercaptoethanol and
different concentrations of GdnHCl at a final protein
concentration of 0.25 mgÆmL
)1
. The unfolding reaction was
carried out at 25 °C for 16 h. For the HPLC analysis, the
reaction was terminated by adding trifluoroacetic acid and
analysed by RP-HPLC on a C4 column. To observe the
time-dependent distribution of the unfolding intermediates
during this process, native HPI was dissolved in the
unfolding buffer (100 m
M

both GSH and GSSG was used as a negative control. The
reaction was carried out at 4 °C overnight. After incuba-
tion, one-fifth of the volume of freshly prepared 0.5
M
sodium iodoacetate solution was added to carboxymethy-
late the free thiol groups of proteins. The carboxymehy-
lation reaction was carried out at room temperature for
5 min. The modified mixture was then analysed by native
PAGE.
Isolation and purification of the scrambled disulfide
isomers of HPI
In the presence of denaturant and thiol catalyst as
indicated above, HPI was converted into the mixture of
native and scrambled disulfide isomers, which existed in a
state of equilibrium. The mixture was adjusted to pH 1.0
with trifluoroacetic acid and separated using RP-HPLC
on a C4 column (Sephasil peptide, ST 4.6/250 mm,
Pharmacia). Unless otherwise indicated, the solvent A
was 0.15% trifluoroacetic acid in water and solvent B
was 60% acetonitrile containing 0.125% trifluoroacetic
acid. The linear elution gradient was 50% B to 80% B
in 30 min with a flow rate of 0.5 mLÆmin
)1
. The
detection wavelength was 280 nm. The partially isolated
disulfide isomers of HPI were further purified by HPLC
on a C8 column (Sephasil peptide, ST 4.6/250 mm,
Pharmacia). The corresponding fraction was collected
and lyophilized.
Disulfide linkage analysis of the intermediates

pH 8.7, 1 m
M
EDTA and 0.2 m
M
2-mercaptoethanol at
final concentration of 0.1 mgÆmL
)1
. The refolding reaction
was carried out at 4 °C. Aliquots of the folding solution
were removed at time intervals and mixed with an equal
volume of 2% trifluoroacetic acid to stop the folding
process. The mixture was then analysed by RP-HPLC on a
C4 column (Sephasil peptide, ST 4.6/250 mm, Pharmacia)
with a linear gradient of 50% B to 80% B in 30 min. The
flow rate was 0.5 mLÆmin
)1
and the detector wavelength
was 230 nm.
Protein analysis
The protein concentration of HPI and the disulfide isomers
were calculated by UV spectroscope using an absorption
constant A
276
(1 cm, 1.0 mg mL
)1
) ¼ 0.65 according to the
reference [36]. The molecular mass of the disulfide isomers
of HPI and the enzyme-digested fragments were measured
by ESI MS. The molecular mass of the mixture of
scrambled isomers was measured by MALDI-TOF MS.

M
dithiothreitol, most of the HPI accumulated as disulfide-
linked aggregates with only a small portion being reduced.
The reduction of HPI by 1 m
M
dithiothreitol is shown in
Fig. 2. The native disulfide bonds of HPI were rapidly
reduced in a collective manner. After 20 min,  95% of the
native HPI had been converted into frHPI. At early
points (2 or 5 min) only a small fraction of intermediates
existed between HPI and frHPI as measured by HPLC. At
10 s or 20 min during the reaction, we could not detect any
visible unfolding intermediates by HPLC. Since there are no
obviously accumulated intermediates during this reducing
process, it is very difficult to study the reductive unfolding
pathway by analysis of intermediates.
Fig. 2. Reductive unfolding of HPI by the addition of 1 m
M
dithio-
threitol in alkaline buffer. The reducing reaction was quenched at dif-
ferent time points, as indicated at the right side of each HPLC
chromatograph. The corresponding peaks of native HPI and reduced
(frHPI) are indicted at the top of the peaks. HPLC conditions are
described in Experimental procedures.
1740 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Native HPI unfolds more readily than PIP using
the disulfide scrambling method
Because it is difficult to study the reductive unfolding of HPI
in the absence of denaturant, we used the disulfide
scrambling method to monitor the HPI unfolding process

native HPI fraction was retained when the concentration
of urea or GdnHCl was 1.0
M
. In contrast, almost 4.0
M
GdnHCl or 8.0
M
urea was needed to reduce the native PIP
fraction to 30%, indicating that PIP is much more able to
maintain its native structure and disulfide bonds than HPI.
Moreover, PIP showed a cooperative unfolding process
with increasing denaturant conditions, while HPI rapidly
lost its native structure even at the lowest concentration of
denaturant.
Fig. 3. Unfolding of HPI in the presence of denaturant and thiol cata-
lyst. Controls for the disulfide scrambling method include the lack of
thiol catalyst and lack of denaturant in the buffer. As both controls
give the same results, only one is shown. Native HPI exists stably in
both control experiments after incubation at 16 °C for 16 h. The three
major peaks containing scrambled disulfide isomers of HPI are desi-
gnated G1, G2 and G3 separately, based on their elution sequence on
HPLC.
Fig. 4. Denaturation curve of HPI and PIP by disulfide scrambling
method. The native fraction retained is the percentage of native HPI
that is not converted into the scrambled isomers. Denaturation was
carried out at 16 °C for 16 h in denaturing buffer containing
0.2 m
M
2-mercaptoethanol and the indicated concentration of
denaturant.

isomers during the disulfide scrambling process
HPI was unfolded by disulfide scrambling in the presence of
6.0
M
GdnHCl and 0.2 m
M
2-mercaptoethanol. The reac-
tion was quenched in a time-course dependent manner by
removing aliquots of the reaction mixture and adjusting the
pH to 1.0 with trifluoroacetic acid, the samples were then
analysed immediately by HPLC (Fig. 6). There were three
main intermediates during the unfolding process of HPI,
designated G1, G2 and G3. There were no other significant
intermediates observable that resembled the partially struc-
tured isomers, such as P1, P2 or P3, found during the
refolding study of HPI [32].The HPLC peaks corresponding
to G1, G2 and G3 were collected, partially purified and
analysed by native PAGE (Fig. 7A). The native gel shows
that the proteins corresponding to peak G1 are much more
homogeneous than those in peaks corresponding to G2 or
G3. To compare the intermediates found here with the
intermediates found during the refolding studies of HPI, a
mixture of intermediates P3 and P4 captured during the
refolding process were used as a marker. Intermediate P3 is
a scrambled disulfide isomer that contains a disulfide bond
B7-A20 and retains a few secondary structure elements,
while intermediate P4 is an unstructured isomer with a
disulfide bond B7-B19 [32]. G1 is similar to P4 in the
mobility on native PAGE. The G2 and G3 isomers contain
mainly the protein fraction similar to G1 plus some

Fig. 5. Disulfide stability of PIP (A) and HPI (B) in redox buffer. Lane
1 is the native protein marker. Lanes 2–8 represent that the ratio of
GSHtoGSSG(m
M
/m
M
). When the disulfide reshuffling reached
equilibrium, the reaction was terminated by addition of IAA to car-
boxymethylate the free thiols. The samples were analysed on 15%
native PAGE and the gel was stained by Coomassie brilliant blue
R250.
1742 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
Fig. 7. Physiochemical properties and disulfide-linkage patterns of the
intermediates. (A)NativePAGE(15%acrylamide)ofthedisulfide
isomers G1, G2 and G3. P34 represents the mixture of intermediates
P3 (upper) and P4 (lower) captured during the oxidized refolding
process of HPI. The frdHPI is the IAA modified reduced/denatured
HPI. (B) Far-UV CD spectra of the disulfide isomers of HPI. G2 is not
shown due to the high degree of similarity with G3. The protein
concentration used was 0.25 mgÆmL
)1
for all the samples. (C) Peptide
mapping of HPI and G1 after digestion by endoproteinase V8. HPI
and isomer G1 were digested with the endoproteinase V8, and the
mixtures were analysed by HPLC on a reversed phase C8 column. The
peptide in peak f5 of G1 has a molecular mass of 2347, which shows
that G1 contains an intra-B chain disulfide bond.
Fig. 6. Time-course unfolding of HPI in the denaturing buffer containing
6.0
M

2-mercaptoethanol in alkaline buffer,
G1 was able to spontaneously reshuffle its non-native
disulfide bonds until native configuration of HPI was
adopted, as shown in Fig. 8. During the refolding process of
G1, only a few accumulated intermediates were observed by
HPLC, among which one major peak corresponded, in
elution time, to the refolding intermediates P2 of HPI. This
indicates that possession of the A20-B19 disulfide bond in
P2 is an important intermediate step during the disulfide
reshuffling of G1 into native HPI. Taken together, the
refolding process of the G1 isomer in Fig. 8 is very similar to
that of P4 as reported previously, further indicating that G1
and P4 are the same intermediates during the unfolding and
refolding process of HPI, respectively. The same inter-
mediate captured in the unfolding and refolding process
suggests that a correlation exists in the molecular mechan-
ism of the unfolding or refolding of HPI.
Discussion
Although the insulin A- and B-chains contain sufficient
structural information for the correct pairing of the disulfide
bonds [33,34], our refolding studies of HPI have shown that
both the A and B chain as well as the C-peptide contain the
information necessary for proper protein folding. Com-
pared with the cooperative, step-by-step formation of
disulfide bonds and native conformation in the folding
pathway of a mini-proinsulin (PIP) which lacks the
C-peptide [35], HPI rapidly adopts a random formation of
all the intramolecular disulfide bonds during an early stage
of the oxidized folding process. As a result of the observed
differences in the molecular folding process of PIP and HPI,

influencing the stability of its disulfide bonds. Due to the
absence of structural information for the C-peptide, we are
not able to determine how the C-peptide interacts with the
insulin A- or B-chains to make the disulfide bonds more
accessible than that PIP. It’s possible that the longer linker
between the B- and A-chains may make the C terminus of
the B-chain more flexible.
There are examples that conformational stability of a
protein can be modulated by changing the lengths of loop or
linker segments. For example, a four-helix bundle protein
Rop has been shown to have inverse correlation between
loop length and stability [43]. The effects of the linkers have
generally been attributed to the increased entropic penalty
associated with fixing the end positions of longer linkers.
Considering the passive role of the linker in proteins like
Rop, we may question whether the role of C-peptide in HPI
refolding is also passive and simply a flexible longer linker.
However, there are at least three examples that have shown
that the 31-amino acid C-peptide does not act as a simple
linker. First, replacing the native C-peptide of HPI with
different short linkers always resulted in lower expression
level and higher disulfide isomers formation in the mam-
malian cells [44], thus the native C-peptide of HPI is
important for its refolding in vivo. Secondly, either alanine
scanning mutagenesis or deletion of three highly conserved
acidic residues (EAED) at the N terminus of the C-peptide
resulted in severe HPI aggregation during refolding [45].
This suggests that the amino acid composition of the
C-peptide is also an important factor for its function.
Finally, the in vitro refolding yield of HPI could easily be

IGF-I with that of HPI, considering the high primary
sequence homology and 3D structure of the insulin
superfamily, we can deduce that the disulfide linkage
pattern of G1 corresponds to the predominant disulfide
isomer IGF-b1 or IGF-b2, which should be [B7-B19,
A6-A11, A7-A20] or [B7-B19, A6-A20, A7-A11]. During
the disulfide scrambling process of proteins, such as PCI
[16], TAP [47], and hirudin [48], the predominant isomer
always contains the disulfide linkage pattern in which the
nearest cysteines in primary sequence pair and form the
beads-form disulfide bonds. Although the isomer IGF-a,
with a Cys47-Cys48 disulfide bond, is adopted to the pattern
of consecutive disulfide linkage, it may be absent from the
folding pathway of fully reduced IGF due to its poor
solubility [49]. There may not be IGFa-like isomers during
the unfolding of HPI because all the isomers are highly
soluble. Maybe this is one of the reasons why IGF-I has a
swap form while HPI/insulin has not.
We have studied the oxidized refolding pathway of HPI
and captured four disulfide isomers as intermediates. P4 was
identified as the most unstructured intermediate and
contains the disulfide bond B7-B19 [32]. In this study, we
found that HPI was converted mainly into a disulfide
isomer G1 during its unfolding in the presence of denaturant
and a trace thiol reagent. The native electrophoresis, CD
spectrum, disulfide linkage analysis and the HPLC beha-
viour of G1 strongly suggest that it is identical to P4. The
identical intermediate captured during the oxidized refold-
ing and disulfide-scrambling unfolding suggests that the
pathway of unfolding and refolding of HPI might be similar

proinsulin and helpful discussion. We are grateful to K. Brazine at the
Dana-Farber Cancer Institute for critical reading of this manuscript.
This work was supported by the grants from the National
Foundation of Natural Science (No.39670179) and Chinese Academy
of Sciences (KJ951-B1-606).
References
1. Shortle, D. (1996) The denatured state (the other half of the fold-
ing equation) and its role in protein stability. FASEB J. 10, 27–34.
2. Rader, A.J., Hespenheide, B.M., Kuhn, L.A. & Thorpe, M.F.
(2002) Protein unfolding: rigidity lost. Proc. Natl Acad. Sci. USA
99, 3540–3545.
3. Hespenheide, B.M., Rader, A.J., Thorpe, M.F. & Kuhn, L.A.
(2002) Identifying protein folding cores from the evolution of
flexible regions during unfolding. J. Mol. Graph. Model. 21,
195–207.
4. Matouschek, A. (2003) Protein unfolding – an important process
in vivo? Curr. Opin. Struct. Biol. 13, 98–109.
5. Li, R., Battiste, J.L. & Woodward, C. (2002) Native-like inter-
actions favored in the unfolded bovine pancreatic trypsin inhibitor
have different roles in folding. Biochemistry 41, 2246–2253.
6. Radford, S.E., Dobson, C.M. & Evans, P.A. (1992) The folding of
hen lysozyme involves partially structured intermediates and
multiple pathways. Nature 358, 302–307.
7. Houry, W.A., Rothwarf, D.M. & Scheraga, H.A. (1995) The
nature of the initial step in the conformational folding of
disulphide-intact ribonuclease A. Nat. Struct. Biol. 2, 495–503.
8. Creighton, T.E. (1984) Disulfide bond formation in proteins.
Methods Enzymol. 107, 305–329.
9. Creighton, T.E. (1979) Intermediates in the refolding of reduced
ribonuclease A. J.Mol.Biol.129, 411–431.

peptide model of insulin folding intermediate with one disulfide.
Protein Sci. 12, 768–775.
21.Weiss,M.A.,Hua,Q.X.,Jia,W.,Chu,Y.C.,Wang,R.Y.&
Katsoyannis, P.G. (2000) Hierarchical protein Ôun-designÕ:insu-
lin’s intrachain disulfide bridge tethers a recognition alpha-helix.
Biochemistry 39, 15429–15440.
22. Guo, Z.Y. & Feng, Y.M. (2001) Effects of cysteine to serine
substitutions in the two inter-chain disulfide bonds of insulin. Biol.
Chem. 382, 443–448.
23. Hua,Q.X.,Chu,Y.C.,Jia,W.,Phillips,N.F.,Wang,R.Y.,Kat-
soyannis, P.G. & Weiss, M.A. (2002) Mechanism of insulin chain
combination. Asymmetric roles of A-chain alpha-helices in disul-
fide pairing. J. Biol. Chem. 277, 43443–43453.
24.Hua,Q.X.,Nakagawa,S.H.,Jia,W.,Hu,S.Q.,Chu,Y.C.,
Katsoyannis, P.G. & Weiss, M.A. (2001) Hierarchical
protein folding: asymmetric unfolding of an insulin analogue
lacking the A7–B7 interchain disulfide bridge. Biochemistry 40,
12299–12311.
25. Hua, Q.X., Gozani, S.N., Chance, R.E., Hoffmann, J.A., Frank,
B.H. & Weiss, M.A. (1995) Structure of a protein in a kinetic trap.
Nat. Struct. Biol. 2, 129–138.
26. Steiner, D.F. (1978) On the role of the proinsulin C-peptide.
Diabetes 27 (Suppl. 1), 145–148.
27. Millican, R.L. & Brems, D.N. (1994) Equilibrium intermediates in
the denaturation of human insulin and two monomeric insulin
analogs. Biochemistry 33, 1116–1124.
28. Bryant,C.,Strohl,M.,Green,L.K.,Long,H.B.,Alter,L.A.,
Pekar, A.H., Chance, R.E. & Brems, D.N. (1992) Detection of an
equilibrium intermediate in the folding of a monomeric insulin
analog. Biochemistry 31, 5692–5698.

mainly controlled by their B-chain/domain. Biochemistry 41,
1556–1567.
1746 C Y. Min et al. (Eur. J. Biochem. 271) Ó FEBS 2004
38. Guo, Z.Y., Shen, L. & Feng, Y.M. (2002) The different energetic
state of the intra A-chain/domain disulfide of insulin and insulin-
like growth factor 1 is mainly controlled by their B-chain/domain.
Biochemistry 41, 10585–10592.
39. Guo, Z.Y., Tang, Y.H., Wang, S. & Feng, Y.M. (2003) Con-
tribution of the absolutely conserved B8Gly to the foldability of
insulin. Biol. Chem. 384, 805–809.
40. Steiner, D.F. (1973) Cocrystallization of proinsulin and insulin.
Nature 243, 528–530.
41. Snell, C.R. & Smyth, D.G. (1975) Proinsulin: a proposed three-
dimensional structure. J.Biol.Chem.250, 6291–6295.
42.Weiss,M.A.,Frank,B.H.,Khait,I.,Pekar,A.,Heiney,R.,
Shoelson, S.E. & Neuringer, L.J. (1990) NMR and photo-CIDNP
studies of human proinsulin and prohormone processing inter-
mediates with application to endopeptidase recognition. Bio-
chemistry 29, 8389–8401.
43. Nagi, A.D. & Regan, L. (1997) An inverse correlation between
loop length and stability in a four-helix-bundle protein. Fold. Des.
2, 67–75.
44. Liu, M., Ramos-Castaneda, J. & Arvan, P. (2003) Role of the
connecting peptide in insulin biosynthesis. J. Biol. Chem. 278,
14798–14805.
45. Chen, L.M., Yang, X.W. & Tang, J.G. (2002) Acidic residues on
the N-terminus of proinsulin C-Peptide are important for the
folding of insulin precursor. J. Biochem. (Tokyo) 131, 855–859.
46. Chang, J.Y., Li, L. & Bulychev, A. (2000) The underlying
mechanism for the diversity of disulfide folding pathways. J. Biol.