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

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

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

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

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

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

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

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-

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

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

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

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

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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Structure of C-peptide oligomeric states J. Lind et al.
3768 FEBS Journal 277 (2010) 3759–3768 ª 2010 The Authors Journal compilation ª 2010 FEBS