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

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

amyloid states

Jesper Lind1,*, Emma Lindahl2,*, Alex Pera´lvarez-Marı´n1,*, Anna Holmlund2, Hans Jo¨rnvall2and Lena Ma¨ler1

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: lena.maler@dbb.su.se

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 demonstrmedi-ated 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 ) by fluorescence technology ( MI:0051 )

l MINT-7975757 : c-peptide (uniprotkb: P01308 ) and c-peptide (uniprotkb: P01308 ) bind ( MI:0407 ) by nuclear magnetic resonance ( MI:0077 )

l MINT-7975840 : c-peptide (uniprotkb: P01308 ) and c-peptide (uniprotkb: P01308 ) bind ( MI:0407 ) by circular dichroism ( MI:0016 )

l MINT-7975708 : c-peptide (uniprotkb: P01308 ) and c-peptide (uniprotkb: P01308 ) bind ( MI:0407 ) by blue native page ( MI:0276 )

l MINT-7975816 : c-peptide (uniprotkb: P01308 ) and c-peptide (uniprotkb: P01308 ) bind ( MI:0407 ) by dynamic 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.

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

Ca2+ levels [4], mitogen-activated protein-kinase

dependent intracellular signaling [5–7] and the

induc-tion of enzyme producinduc-tion [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 mid-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

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 Mg2+ and Ca2+ in the concentration range 1–10 mm High concentrations of

Mg2+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 Mg2+ did not affect oligomer distribu-tion (Fig 1B) Ca2+ 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 Ca2+and Mg2+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.

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 m2Æs)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.

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 l M of ThT in 10 m M 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.

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

6 mm), 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 m M 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 m M SDS; grey open circle,

2 m M SDS; grey solid square, 3 m M SDS; grey open square, 6 m M

SDS; grey solid triangle, 10 m M SDS; black cross, 15 m M SDS.

Inset shows C-peptide SDS independent behavior in 10 m M sodium

phosphate buffer (pH 7.3) at 20 C SDS concentrations: buffer,

2 m M SDS and 15 m M 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).

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.

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 37C for 15 min before

were prepared in distilled water Samples consisting of

b-C-peptide (100 lm) were incubated with 1 or 10 mm

MgCl2 or CaCl2 at 37C 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 and 50–300 mm NaCl or 50 mm formamide

were incubated at 37C 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

(http://rsb.info.nih.gov/ij/)

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)

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,

10 min of drying with temperature stabilization at 20C,

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

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 Instruinstru-ments, Malvern, UK) at

20C 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

frequency of 400 MHz Increasing amounts of SDS were added to samples containing 0.5 mm C-peptide in either

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

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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