Tài liệu Báo cáo khoa học: Structural features of proinsulin C-peptide oligomeric and amyloid states pptx

Structural features of proinsulin C-peptide oligomeric and
amyloid states
Jesper Lind
1,
*, Emma Lindahl
2,
*, Alex Pera
´
lvarez-Marı
´n
1,
*, Anna Holmlund
2
, Hans Jo
¨
rnvall
2
and
Lena Ma
¨
ler
1
1 Department of Biochemistry and Biophysics, Center for Biomembrane Research, The Arrhenius laboratory, Stockholm University, Sweden
2 Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden
Keywords
C-peptide; diabetes; oligomer; spectroscopy;
structure
Correspondence
L. Ma
¨
ler, Department of Biochemistry and

Biophysics, Center for Biomembrane
Research, The Arrhenius laboratory,
Stockholm University, SE-106 91
Stockholm, Sweden
Fax: +46 8 155597
Tel: +46 8 162448
E-mail:
Present Address
Department of Molecular and Cell Biology,
Harvard University, Cambridge MA 02138,
USA
*These authors contributed equally to this
work
(Received 27 May 2010, revised 8 July
2010, accepted 13 July 2010)
doi:10.1111/j.1742-4658.2010.07777.x
The formation and structure of proinsulin C-peptide oligomers has been
investigated by PAGE, NMR spectroscopy and dynamic light scattering.
The results obtained show that C-peptide forms oligomers of different
sizes, and that their formation and size distribution is altered by salt and
divalent metal ions, which indicates that the aggregation process is medi-
ated by electrostatic interactions. It is further demonstrated that the size
distribution of the C-peptide oligomers, in agreement with previous studies,
is altered by insulin, which supports a physiologically relevant interaction
between these two peptides. A small fraction of oligomers has previously
been suggested to be in equilibrium with a dominant fraction of soluble
monomers, and this pattern also is observed in the present study. The addi-
tion of modest amounts of sodium dodecyl sulphate at low pH increases
the relative amount of oligomers, and this effect was used to investigate the
details of both oligomer formation and structure by a combination of bio-

physical techniques. The structural properties of the SDS-induced oligo-
mers, as obtained by thioflavin T fluorescence, CD spectroscopy and IR
spectroscopy, demonstrate that soluble aggregates are predominantly in
b-sheet conformation, and that the oligomerization process shows charac-
teristic features of amyloid formation. The formation of large, insoluble,
b-sheet amyloid-like structures will alter the equilibrium between mono-
meric C-peptide and oligomers. This leads to the conclusion that the oligo-
merization of C-peptide may be relevant also at low concentrations.
Structured digital abstract
l
MINT-7975828: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind
(
MI:0407)byfluorescence technology (MI:0051)
l
MINT-7975757: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind
(
MI:0407)bynuclear magnetic resonance (MI:0077)
l
MINT-7975840: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind
(
MI:0407)bycircular dichroism (MI:0016)
l
MINT-7975708: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind
(
MI:0407)byblue native page (MI:0276)
l
MINT-7975816: c-peptide (uniprotkb:P01308) and c-peptide (uniprotkb:P01308) bind
(
MI:0407)bydynamic light scattering (MI:0038)
Abbreviations

ATR, attenuated total reflectance; b-C-peptide, biotinylated human C-peptide; CMC, critical micelle concentration; DLS, dynamic light
scattering; ThT, thioflavin T.
FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS 3759
Introduction
C-peptide is derived from most of the proinsulin seg-
ment in between the B and A chains of insulin [1] and
has an important structural role in the proper folding
and disulfide bonding in insulin [2]. After proinsulin
cleavage, it is released to the blood together with insu-
lin in equimolar amounts. The 31-residue peptide has
also been shown to have biological effects of its own at
three principally different locations: at the cell surface,
intracellularly and extracellularly. At the cell surface, it
binds to cell membranes [3], where it has an effect on
Ca
2+
levels [4], mitogen-activated protein-kinase
dependent intracellular signaling [5–7] and the induc-
tion of enzyme production [8]. Regarding internaliza-
tion, C-peptide enters into different cells [9–11], and
into nucleoli, with intracrine effects similar to a growth
factor affecting ribosomal RNA synthesis [11]. Finally,
it has been demonstrated that, extracellularly, C-pep-
tide is involved in the disaggregation of insulin, increas-
ing insulin bioavailability by monomerization [12,13].
C-peptide itself has been shown to adopt unordered
structures in aqueous solutions, although it has some
defined structural segments and is not influenced fur-
ther by the presence of negatively-charged lipid vesicles
[7,14,15]. Similar to many peptides, however, it has a

propensity to form a a-helical structure in the presence
of trifluoroethanol [15]. In addition, molecular dynam-
ics simulations propose turn-like motifs in the mid-
region and in the C-terminal region [16].
The ability of peptides and proteins to self-associate
has been recognized in several diseases, including
Alzheimer’s disease, amyotrophic lateral sclerosis and
type II diabetes [17,18]. The observation that self-asso-
ciating peptides and proteins are at the core of several
neurodegenerative diseases has led to a massive effort
aiming to understand the physiologically relevant
structures and mechanisms involved in this process.
Early studies on proinsulin and insulin behavior in
solution revealed self-associating properties [19–21]
and, as a result, insulin is found to form zinc-induced
hexamers in vivo with deferred bioactivity. Other stud-
ies revealed that insulin also can form amyloid-like
structures in vitro [22], with proinsulin being less sus-
ceptible to fibrillation than insulin alone [23].
The oligomeric states of several endogenous peptides
have been shown to be of relevance with respect to
their physiological function. Recently, it was demon-
strated, under a wide variety of conditions, including
at different pH levels and concentrations, that a small
fraction of C-peptide exists as oligomers, as shown
both by MS and gel electrophoresis [13], as well as by
surface plasmon resonance [12]. This lead us to exam-
ine the structure and physical properties of these states
further. Peptides and protein oligomers have been
extensively detected and studied using techniques such

as size exclusion chromatography, light scattering, elec-
tron microscopy, MS, gel electrophoresis and a wide
range of spectroscopic techniques.
Suitable methods for investigation of the secondary
structure and morphology of such structures, however,
require the presence of large amounts of the oligomeric
state, which does not appear to be the native condition
for C-peptide [13]. The structural features of the aggre-
gation properties of the amyloid precursor protein, as
well as of the opioid peptide dynorphin, were able to
be investigated by trapping stable oligomers through
interaction with modest amounts of a detergent (SDS)
[24,25]. In those studies, it was observed that low con-
centrations of SDS have the ability to mimic the neces-
sary conditions for the formation of aggregated
species, whereas higher concentrations [well above the
critical micelle concentration (CMC)] instead promote
the formation of a-helical structures, protected from
aqueous solvent [26]. Hence, this appears to be a good
model for performing structural studies. In the present
study, we therefore used SDS to structurally character-
ize the oligomerization process of C-peptide and ana-
lyzed the formation of oligomers and their secondary
structure by complementary methods, including the
detection of C-peptide oligomers by PAGE electropho-
resis and spectroscopic techniques. The results
obtained demonstrate that C-peptide forms different
oligomeric states with defined secondary structures in
solution, and we show that this process is mediated
through specific interactions, involving ionic strength

and pH. The equilibrium between monomeric C-pep-
tide and oligomers may be altered by factors such as
local pH and local peptide concentrations in vivo. Con-
version of C-peptide into insoluble aggregates may fur-
ther affect this equilibrium. The results of the present
study also show that C-peptide oligomerization is
affected by the presence of insulin, which supports the
previous conclusions [12,13] that insulin and C-peptide
have physiologically relevant interactions other than
those taking place during synthesis and secretion in the
pancreas.
Results
C-peptide forms oligomers
To confirm that C-peptide forms oligomeric structures,
solutions of biotinylated C-peptide were analyzed by
Structure of C-peptide oligomeric states J. Lind et al.
3760 FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS
PAGE and immunoblotting (anti-biotin). The results
obtained show that C-peptide forms oligomers that
appear to increase with time (Fig. 1A), in agreement
with previous observation under native conditions [13].
Also in agreement with the previous results [13],
monomeric C-peptide is not detected in the staining
used the present study. This implicates that the stain-
ing results may not represent more than a fraction of
C-peptide undergoing oligomerization. In most experi-
ments, the presence of very large aggregates was also
observed.
C-peptide properties have been reported to be influ-
enced by metal ions [27] and we therefore investigated

the effect of different ions on oligomer formation.
Biotinylated human C-peptide (b-C-peptide) was
incubated with solutions of Mg
2+
and Ca
2+
in the
concentration range 1–10 mm. High concentrations of
Mg
2+
appeared to reduce the formation of larger olig-
omeric species (15–30 kDa), whereas a strong band
corresponding to peptide dimers is apparent. Low con-
centrations of Mg
2+
did not affect oligomer distribu-
tion (Fig. 1B). Ca
2+
was also observed to have some
effect on oligomer distribution, with the most signifi-
cant effect being a decrease in medium-order oligomers
(15–30 kDa). In conclusion, divalent ions were seen to
affect C-peptide oligomer formation.
In previous studies, C-peptide could disaggregate
insulin oligomers and, vice versa, insulin could disag-
gregate C-peptide oligomers [12,13]. In the present
study, we found that, at 10 lm of insulin, the presence
Fig. 1. Oligomer formation of proinsulin
C-peptide is affected by metals and insulin.
Prosinsulin C-peptide oligomer formation as

a function of time (A). C-peptide was incu-
bated for the indicated time and analyzed
under native conditions. Oligomer distribu-
tion of 100 l
M proinsulin C-peptide in the
presence of divalent Ca
2+
and Mg
2+
ions
under native conditions (B), and of 100 l
M
C-peptide in the presence of insulin (C) and
of 100 l
M C-peptide in the presence of NaCl
and formamide (D) under native conditions.
J. Lind et al. Structure of C-peptide oligomeric states
FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS 3761
of medium-order C-peptide oligomers (15–30 kDa)
appears to be reduced, although it is difficult to judge
by what extent at higher concentrations of insulin
(Fig. 1C). An interaction is therefore likely between
C-peptide and insulin, leading to an effect on the oli-
gomer. The effect of NaCl and formamide on oligomer
formation was also tested. NaCl breaks electrostatic
interactions, whereas formamide breaks hydrophobic
ones. The addition of 50–150 mm NaCl reduced the
oligomer formation of C-peptide (Fig. 1D) and form-
amide had no effect (Fig. 1D). The combined results
therefore indicate that the oligomerization process is

related to electrostatic interactions, and that insulin
affects the oligomer distribution. Prolonged incubation
of solutions only containing C-peptide increased the
presence of higher-order oligomers. This supports the
conclusion that small amounts of oligomers exist in
equilibrium with a much larger fraction of peptide
monomers [13], and that the formation of aggregates
may shift the equilibrium towards larger amounts of
oligomers over time.
NMR and dynamic light scattering (DLS) reveal
the presence of large aggregates
The size of the C-peptide aggregates were initially
investigated by recording 1D NMR spectra and per-
forming pulsed-field gradient diffusion NMR. Through
repeated measurements, the diffusion constant of
C-peptide in solution (500 lm) was determined to
be 1.76 · 10
)11
m
2
Æs
)1
. By relating this value to a
calibrated version of Stoke–Einsteins relationship, a
molecular weight of 3060 ± 90 Da is derived [28]. This
value is very close to the theoretical molecular weight
of the monomeric C-peptide (3020.3 Da), which indi-
cates that C-peptide is mainly monomeric, even at the
high peptide concentration used in the NMR experi-
ment. This result indicates, in agreement with the gel

electrophoresis results, that only a small fraction of the
peptide had formed oligomers, and that the population
of oligomers is below the detection limit in the NMR
measurements.
Increasing amounts of SDS was added to solutions
of 500 lm proinsulin C-peptide at pH 8 and pH 3.2.
At pH 8, neither the diffusion rate, nor the signal-to-
noise ratio for the peptide signals in the spectrum is
severely affected by the addition of detergent (data not
shown). At pH 3.2, SDS has a completely different
effect on C-peptide solutions. The diffusion coefficient
for the peptide was only slightly altered by adding
SDS, although the signal intensity (normalized signal-
to-noise ratio) for the peptide decreased significantly
with increasing amounts of SDS. The signal reduction
indicates that a substantial part of the peptide partici-
pates in large (NMR-invisible) oligomer complexes
(Fig. 2A). Therefore, the measured diffusion coeffi-
cients for C-peptide in SDS solution represent the
remaining population of NMR-visible monomers
because the increasing fraction of oligomers (with
increasing SDS concentration) does not result in visible
NMR signals. Hence, the only way that we could
directly detect the formation of large oligomers by
NMR was by a loss of signal intensity (Fig. 2A). Simi-
lar observations were previously made for aggregating
Fig. 2. C-peptide forms large oligomers. Normalized signal-to-noise
ratios for resonances in the
1
H-NMR spectrum of the proinsulin

C-peptide, (0.9 p.p.m., squares) and for acetate buffer (2.0 p.p.m.,
circles) as a function of SDS concentration at pH 3.2 (A). Size distri-
bution of prosinsulin C-peptide oligomers measured by DLS in the
presence of 0, 0.5, 1.0, 1.5, 3 and 10.0 m
M SDS at pH 3.2 (B). The
size is expressed as the hydrodynamic radius.
Structure of C-peptide oligomeric states J. Lind et al.
3762 FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS
peptides, such as amyloid precursor protein [24]. As a
control, the signal intensity of acetate (at 2 p.p.m.) was
also monitored as a function of the SDS content
(Fig. 2A) and, as expected, no significant effect on the
peak intensity is seen at low or moderate SDS concen-
trations, which means that SDS specifically induces the
oligomerization of C-peptide, and does not alter other
conditions.
In summary, C-peptide is predominantly a monomer
in buffer solutions at low pH and, in the presence of
modest amounts of SDS, the peptide oligomerizes into
large complexes but with a remaining monomer popu-
lation. At increased SDS concentration, the large com-
plexes are dissolved by the detergent.
C-peptide aggregates were also monitored with DLS
(Fig. 2B). With no SDS present in the sample, mea-
surements showed a single population of monomers
with a hydrodynamic radius of approximately 16 A
˚
,
which is in agreement with the results of the NMR.
The addition of SDS to the C-peptide sample leads to

formation of larger objects, even at an SDS concentra-
tion of only 500 lm, which is well below the CMC.
The relative sizes of the oligomers increase gradually
with higher SDS concentrations. By contrast to the
NMR experiments, in which only small species can be
detected, the monomer state cannot be discerned
by light scattering in the presence of the much larger
oligomer complexes because of the strong size depen-
dency of this method. Despite an equilibrium time of
24 h, the conditions most likely do not represent equi-
librium, and hence any conclusions about the calcu-
lated population distributions cannot be made. The
DLS experiments, however, do confirm the formation
of C-peptide oligomers in the presence of SDS. They
also confirm that the distribution of oligomers must
include large species (such as those observed in the gel
electrophoresis experiments) because the average size
from the DLS measurements corresponds to a hydro-
dynamic diameter of approximately 10 nm, which is
too large to indicate only dimers or trimers.
C-peptide forms amyloid-like aggregates
Thioflavin T has been used to detect aggregates of sev-
eral amyloidogenic peptides and proteins [29] and was
also used in a previous study of C-peptide [13]. We
now performed experiments with 500 lm C-peptide at
pH 3.2 in the presence of 15 lm thioflavin T (ThT)
(Fig. 3). Increasing amounts of SDS were added to the
samples to detect the fluorescence increase of ThT
when oligomers or aggregated forms appeared. The
maximum in ThT fluorescence intensity was observed

at 2 mm SDS, following a sigmoidal trend as the SDS
concentration increased. The midpoint for this sigmoid
was at 1.2 mm. Subsequent detergent titration steps
decreased the ThT fluorescence and, at 15 mm SDS,
almost to the initial intensity observed without SDS.
As a control, the same experiment was performed in
the absence of peptide, in which case virtually no
changes in fluorescence intensity were observed
(Fig. 3).
Secondary structure of C-peptide oligomeric
states
To monitor the structural transitions accompanying
the SDS-induced aggregation observed with the fluo-
rescence and DLS experiments, a combination of CD
and FTIR spectroscopy was used.
First, the effect of increasing concentrations of SDS
on C-peptide was investigated by CD spectroscopy
(Fig. 4). At pH 7.3, no induced secondary structure
was detected, and C-peptide was seen to be in a ran-
dom coil conformation at all SDS concentrations
(Fig. 4A, inset). To determine whether the charges in
the peptide were relevant, the same experiments were
carried out at pH 3.2 (below the theoretical isoelectric
point of the peptide; Fig. 4A). At this acidic pH,
a clear transition from random coil to b-sheet structure
was observed with increasing SDS concentration. The
b-sheet contribution was maximal at 2 mm SDS
(Fig. 4B). As the SDS concentration increased to and
Fig. 3. SDS induced oligomerization of C-peptide monitored by ThT
fluorescence. Tht fluorescence intensity at 480 nm for a solution of

500 l
M proinsulin C-peptide and 15 lM of ThT in 10 mM sodium
acetate buffer (pH 3.2) in the presence of the indicated amount of
SDS (open circles). As a control, measurements were also per-
formed with a solution containing Tht only (pH 3.2) in the presence
of the indicated amount of SDS (filled circles). Measurements were
performed at 20 °C.
J. Lind et al. Structure of C-peptide oligomeric states
FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS 3763
above the CMC, the b-sheet content decreased, yield-
ing a more a-helix-like spectrum at 15 mm SDS
(Fig. 4). Interestingly, with higher concentrations of
SDS (pH 3.2), part of the signal appears to disappear
from the spectrum, consistent with the NMR observa-
tions and previous studies showing that the presence
of larger aggregates leads to a disappearing CD signal
[24].
We then turned to solid-state attenuated total reflec-
tance (ATR)-IR spectroscopy to analyze a film of dry
C-peptide. The amide I region of the spectrum has been
widely used to assess the secondary structure of pep-
tides, including in aggregation processes [30,31], and
this region of the spectrum was utilized as an indicator
for a structural transition. To determine the secondary
structure transition, 1638 cm
)1
was assumed as the
threshold between random coil and b-sheet structure
[32]. Higher wavenumber values are dominated by ran-
dom coil and a-helix, whereas lower wavenumber val-

ues are attributed to b-sheet structure only. In the
absence of SDS, C-peptide shows an amide I band cen-
tered at 1638 cm
)1
(Fig. 5A). As the SDS concentra-
tion increases, the amide I maximum shifts to lower
wavenumbers and a shoulder becomes prominent at
1618 cm
)1
. At the highest SDS concentrations (above
6mm), the maximum shifts back to higher wavenumber
values, indicating the loss of b-sheet and the onset of
a-helix structure formation. To visualize the trend in
the formation of b-sheet as a function of increasing
SDS concentrations, the ratio between the bands at
1618 and 1638 cm
)1
is plotted in Fig. 5B. The b-sheet
contribution was most significant when the SDS
concentration was 1–6 mm, reaching a maximum at
2–4 mm, which is qualitatively in agreement with the
results of the CD spectroscopy. At higher SDS : peptide
ratios, the b-sheet contribution dropped, again in agree-
ment with the solution-state CD spectroscopy results,
reaching the same level as that in the absence of SDS.
In conclusion, we find that the formation of oligo-
meric species is accompanied by a structural transition
from a largely random coil C-peptide structure to pre-
dominantly b-sheet, and that the b-sheet structure dis-
appears with SDS concentrations around or above the

CMC.
Discussion
In the present study, we have detected and examined
oligomer structures of proinsulin C-peptide, which
appear to be formed by electrostatic interactions. We
observe that high concentrations of salt reduce the size
of the oligomers, whereas formamide, which breaks
hydrophobic interactions, has no effect (Fig. 1). Fur-
thermore, divalent metal ions also affect the oligomeri-
zation. A variety of different species are formed, as
demonstrated by gel electrophoresis. The formation of
the aggregates is time-dependent and longer incubation
time results in larger aggregates. This indicates that
amyloidogenic species are formed (Fig. 1), which alter
the equilibrium between the monomeric C-peptide and
the oligomers.
To investigate the structural features of the aggre-
gates, or oligomers, a relatively high concentration of
Fig. 4. SDS secondary structure induction. (A) CD spectra of
500 l
M proinsulin C-peptide in 10 mM sodium acetate buffer (pH
3.2) at 20 °C in the presence of increasing SDS concentrations:
black open square, buffer; grey solid circle, 0.5 m
M SDS; grey open
triangle, 1 m
M SDS; black solid star, 1.5 mM SDS; grey open circle,
2m
M SDS; grey solid square, 3 mM SDS; grey open square, 6 mM
SDS; grey solid triangle, 10 mM SDS; black cross, 15 mM SDS.
Inset shows C-peptide SDS independent behavior in 10 m

M sodium
phosphate buffer (pH 7.3) at 20 °C. SDS concentrations: buffer,
2m
M SDS and 15 mM SDS. (B) Plot of the mean residual molar
ellipticity at 195 nm for the C-peptide SDS titration in sodium ace-
tate buffer (pH 3.2).
Structure of C-peptide oligomeric states J. Lind et al.
3764 FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS
peptide is required. It was demonstrated in an previ-
ous study, however, that C-peptide indeed undergoes
conversion from monomer to oligomer states at a
wide range of conditions, including concentration [13].
Hence, the results obtained in the present study are
likely comparable to those seen under conditions
with low concentrations more resembling an in vivo
situation. Remarkably, as noted earlier at lower
concentrations, it appears that, even at the higher
concentrations used in the present study, the dominant
fraction of C-peptide is momomeric but in equilibrium
with a population of oligomers. Our structural analy-
ses only detected the presence of oligomers upon the
addition of modest amounts (relative to the peptide
concentration) of SDS, indicating that the equilibrium
between nomomers and oligomers remains.
We find that the amount of oligomers formed is
enhanced by the addition of modest amounts of SDS
to solutions of C-peptide. Previous studies have indi-
cated that SDS promotes the formation of oligomers
in different peptides [24], and we used this effect to
investigate the structural features of the oligomers.

These oligomers are predominantly b-sheet, as demon-
strated by both CD spectroscopy and ATR-IR spec-
troscopy (Figs 4 and 5). Interestingly, this SDS
induced oligomer formation is very pH-dependent. At
a pH close to the isoelectric point of the acidic peptide
(predicted pI of approximately 3) oligomers are
formed, whereas, at pH 7.3, no structure in the peptide
is observed. This again agrees with the assumption that
the oligomer formation is electrostatic in nature.
The NMR solution structure of proinsulin C-peptide
in aqueous solution is essentially random. Weak ten-
dencies to form b-turns in trifluoroethanol solution
have been suggested [15], whereas CD spectroscopy
indicates that the peptide becomes helical in this
solvent [14]. Furthermore, interactions with lipid vesi-
cle bilayers do not result in any membrane-induced
structure conversion in C-peptide, which indicates that
physiological effects of C-peptide are most likely not
mediated by direct membrane interactions [14]. These
previous findings suggest that the structure conversion
to the oligomers seen in the present study is not medi-
ated by membrane (lipid) interactions but rather by
electrostatic interactions, as indicated by salt and pH
effects. This result is very similar to those seen for
other acidic and amyloidogenic peptides, such as the
Alzheimer amyloid b-peptide, which has many com-
mon features with C-peptide [33], and insulin. It is well
known that insulin forms oligomeric states and amy-
loid fibrils as a function of pH and ionic strength
[19,34–37]. C-peptide has also been demonstrated to be

likely to form oligomers under conditions more similar
to situations in vivo, including sub-lm concentrations
[12,13]. Local concentrations of C-peptide and local
pH effects may shift the equilibrium between C-peptide
monomer and oligomer species, promoting the forma-
tion of insoluble amyloid-like structures. If amyloid
structures are formed anywhere in vivo, this equilib-
rium may further shift rapidly.
In summary, we have shown that electrostatic inter-
actions promote the formation of C-peptide b-sheet
Fig. 5. Solid-state secondary structure of C-peptide induced by
SDS. (A) Films of proinsulin C-peptide in the presence of increasing
SDS concentrations were dried on the ATR diamond surface and
FTIR spectra were acquired. The corresponding SDS : peptide
ratios are indicated. The dashed lines indicate the threshold
between random coil and b-sheet structures (1638 cm
)1
) and a rep-
resentative b-sheet wavenumber (1618 cm
)1
). (B) The b-sheet :
random coil ratio is plotted to illustrate the b-sheet content at each
SDS : peptide ratio.
J. Lind et al. Structure of C-peptide oligomeric states
FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS 3765
oligomers, and that these oligomers can form amyloid
structures. In a previous study of C-peptide oligomers
[13], it was shown that they are formed under a wider
set of conditions (low concentration, weakly acidic or
basic pH), but in modest amounts, which appear to be

in equilibrium with a much larger fraction of mono-
mers. In the present study, we have characterized the
structural features of the C-peptide oligomerization
process, and we find that this oligomerization process
has the characteristic features of amyloid formation.
Even if the equilibrium between monomer species and
oligomer states is such that C-peptide is mainly mono-
meric, small amounts of amyloid formation will alter
this equilibrium.
Experimental procedures
Native and SDS/PAGE
Stock solutions of 400 lm b-C-peptide (GenScript Corpora-
tion, Piscataway, NJ, USA) were prepared in 20 mm Hepes
buffer (pH 7.9), diluted to concentrations in the range
25–200 lm and incubated at 37 °C for 15 min before analy-
sis by SDS ⁄ PAGE and native PAGE. Stock solutions of
20 mm MgCl
2
, and CaCl
2
(Merck, Darmstadt, Germany)
were prepared in distilled water. Samples consisting of
b-C-peptide (100 lm) were incubated with 1 or 10 mm
MgCl
2
or CaCl
2
at 37 °C for 30 min. Samples containing
10, 50, 100, 200 and 400 lm of human insulin (Actrapid;
NovoNordisk, Bagsværd, Denmark) were incubated with

b-C-peptide at 37 °C for 15 min. Samples containing
b-C-peptide and 50–300 mm NaCl or 50 mm formamide
were incubated at 37 °C for 15 min.
Tris-glycine native sample buffer (·2) was added to the
samples for native PAGE and 20 lL samples were sepa-
rated on 16% Tris glycine gels (Invitrogen, Carlsbad, CA,
USA). The gels were transferred to poly(vinylidenedifluo-
ride) membranes that were probed with a streptavidin anti-
body (Calbiochem, San Diego, CA, USA). Analysis of
band intensities was performed using the imagej software
( />CD spectroscopy
CD measurements were performed for samples containing
500 lm C-peptide at pH 7.3 and 3.2 (10 mm sodium phos-
phate buffer and 10 mm sodium acetate buffer, respectively)
at 20 °C. Increasing amounts of SDS (from a 1 m stock
solution) were added to the samples. CD spectra were
acquired using a quartz cuvette with a 0.01 mm optical
path length with an Applied Photophysics Chirascan spec-
trometer (Applied Photophysics, Leatherhead, UK). Spec-
tra were collected in the range 185–250 nm with a 0.5 nm
step increment. The detection response time was 0.5 s at
1 nm bandwidth and three scans were collected and aver-
aged for each experiment.
ATR-IR spectroscopy
Aliquots of samples were taken from each of the samples
used for CD spectroscopy and dried over the ATR dia-
mond surface of a Bruker Vortex spectrometer (Bruker,
Ettlingen, Germany) using a gentle N
2
stream. After

10 min of drying with temperature stabilization at 20 °C,
500 scans were collected and averaged at 4 cm
)1
. The
amide I region (approximately 1700–1600 cm
)1
) was used
for the analysis of the secondary structure of the peptide.
ThT fluorescence
Fluorescence measurements for samples containing 500 lm
peptide in 10 mm acetate buffer, pH 3.2, and 15 lm ThT
were made on a Jobin-Yvon Fluoromax spectrofluorometer
(HORIBA Jobin Yvon Inc., Edison, NJ, USA) using a
1 cm quartz cuvette with gentle stirring. All measurements
were performed at 20 °C. Increasing amounts of SDS were
added to the samples. As a control, measurements were
performed both in the absence and presence of peptide.
ThT fluorescence was excited at 450 nm (1 nm slit width)
and single wavelength emission measurements at 483 nm
(1 nm slit width) were performed with a 1 s detector
response time.
DLS
All DLS measurements were recorded on a Zetasizer instru-
ment (Nano ZS; Malvern Instruments, Malvern, UK) at
20 °C using a standard disposable polystyrene cuvette of
1 cm path length. Increasing amounts of SDS (from a 1 m
SDS stock solution) were added to samples containing
0.5 mm C-peptide dissolved in 10 mm sodium acetate buffer
(pH 3.2). The samples were equilibrated for 24 h prior to
every measurement. Scattering data were collected as an

average of ten scans collected over 120 s. The data were
processed in accordance with the manufacturer’s software
(dts; Malvern Instruments) and presented as scattering
intensity autocorrelation decays. The Stoke–Einstein rela-
tionship, together with refractive indices and temperature
corrected viscosities provided by the dts software, was used
to calculate the hydrodynamic radius of the aggregates.
NMR spectroscopy
All NMR experiments were carried out at a temperature of
25 °C on a Bruker Avance spectrometer equipped with
a broad band inverse probe operating at a
1
H Larmor
frequency of 400 MHz. Increasing amounts of SDS were
added to samples containing 0.5 mm C-peptide in either
Structure of C-peptide oligomeric states J. Lind et al.
3766 FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS
10 mm sodium acetate buffer (pH 3.2) or 10 mm phosphate
buffer (pH 8.0). The samples were equilibrated for 24 h
prior to every measurement. NMR spectral intensities in
1D
1
H-NMR spectra were recorded as a function of the
SDS concentration, using a 90° excitation pulse followed by
excitation sculpting water suppression [38] and data were
collected as an average over 64 scans. Diffusion coefficients
were measured using the pulse-field gradient spin-echo
experiment with a fixed diffusion time and bipolar pulsed
field gradients increasing linearly over 32 steps [39,40].
Measured diffusion coefficients were related to a molecu-

lar weight via a modified version of the Stoke–Einstein
relationship [28].
Acknowledgements
We thank Andreas Barth for access to the FTIR spec-
trometer. This work was supported by grants from the
Swedish Research Council, The Carl Trygger Founda-
tion, The Magnus Bergvall Foundation and from
European Union (Marie Curie Action PIOF-GA-2009-
237120 to A. P. -M.).
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