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: 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 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 (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) 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.). References 1 Steiner DF (1967) Evidence for a precursor in the biosynthesis of insulin. 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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 . Structural features of proinsulin C-peptide oligomeric and amyloid states Jesper Lind 1, *, Emma Lindahl 2, *,. 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