Vortex-Induced Amyloid Superstructures of Insulin and Its Component

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Vortex-Induced Amyloid Superstructures of Insulin and Its Component A and B Chains Viktoria Babenko,† Marcin Piejko,‡,§ Sławomir Wójcik,† Paweł Mak,‡ and Wojciech Dzwolak*,† †

Department of Chemistry, University of Warsaw, Pasteura 1, 02-093 Warsaw, Poland Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Cracow, Poland

‡

S Supporting Information *

ABSTRACT: Insulin is an amyloid-forming polypeptide built of two disulfide-linked chains (A and B), both themselves amyloidogenic. An interesting property of insulin is that agitation strongly influences the course of its aggregation, resulting in characteristic chiral superstructures of amyloid fibrils. Here, we investigate the self-assembly of these superstructures by comparing the quiescent and vortex-assisted aggregation of insulin and its individual A and B chains in the presence or absence of reducing agent tris(2-carboxyethyl)phosphine (TCEP). Our study shows that only the B chain in the presence of TCEP is converted into aggregates with morphology (according to atomic force microscopy) and optical activity (manifested as an extrinsic Cotton effect induced in bound thioflavin T) characteristic of amyloid superstructures that are normally formed by insulin in the absence of TCEP. In contrast to more rigid B-peptide fibrils, elongated aggregates of the A peptide become amorphous upon agitation. Moreover, the aggregation of equimolar mixture of both peptides does not produce highly ordered entities. Our results suggest that the dynamics of the B chain are the driving force for the assembly of superstructures, with the A chain being complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin.

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INTRODUCTION Amyloid fibrils are linear β-sheet-rich aggregates of misfolded protein molecules forming spontaneously both in vivo and in vitro. In the former case, amyloidogenesis has often been linked to fatal degenerative disorders such as Alzheimer’s and Parkinson’s diseases.1 Nowadays, early aggregates rather than mature amyloid fibrils are thought to be most pathogenic. Meanwhile, several examples of purposeful in vivo formation of biologically functional amyloid fibrils have been recognized,2 and in-vitro-synthesized amyloid-like fibrils are being employed in material science and nanotechnology.3,4 Despite the biomedical importance of amyloid fibrils, the current understanding of their structure and the processes leading to their formation is far from satisfying, mainly because of the noncrystallizable character and polymorphism of fibrils as well as the irreversibility of amyloidogenic self-assembly. Thus, in vitro biophysical studies on model amyloidogenic proteins and peptides are of great value. Insulin is one of the most interesting in vitro model amyloidogenic precursors,5−8 although its aggregation may also occur in vivo, subcutaneously at sites of frequent injections.9 In this newfound role, the hormone has been proven to be very useful in studies on polymorphism,10−15 seeding properties,16,17 thermodynamics,18−20 and aggregation pathways21 of amyloid fibrils. Certain structural peculiarities of the covalent structure of insulin have attracted much interest in the amyloid context. Namely, the insulin monomer is built of two polypeptide chains: a 21 amino acid residue long A chain © 2013 American Chemical Society

and a 30 residue long B chain, both connected by two disulfide bonds, whereas a third intrachain S−S bond links two more cysteine residues of the shorter peptide (Figure 1). In both the A and B chains, amyloidogenic regions have been mapped.22,23

Figure 1. Amino acid sequence of bovine insulin. Disulfide bridges are marked with gray dotted lines. The residues of two amyloidogenic regions are marked with filled circles (according to refs 22 and 23). Received: February 17, 2013 Revised: March 16, 2013 Published: April 1, 2013 5271

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and dimethylaminobenzene rings affects the quantum yield of ThT fluorescence. More importantly, the twisting induces optical activity because all ThT rotamers are chiral except for those that are perfectly planar or twisted at the right angle.37 Therefore, an extrinsic Cotton effect (or ICD) in amyloidbound ThT reflects the chirality of dye-binding amyloid surface moieties being imprinted on ThT. The agitation of bovine insulin at 60 °C results in superstructures with uniformly negative ICD signals, so-called −ICD fibrils. However, aggregation at 35 °C favors superstructures with positive ICD signals (+ICD fibrils).12,13 In the intermediate temperature range, the ICD sign is stochastically determined (i.e., microscopic fluctuations lead to the conversion of macroscopic amounts of insulin into aggregates with predominantly positive or negative ICDs, the key characteristics of the chiral bifurcation phenomena observed for phase transitions12,13). Although new and fascinating aspects of the chirality of insulin amyloid fibrils continue to emerge,41,43 so far mechanisms underlying the formation of ±ICD-type superstructures of insulin fibrils are poorly understood. Likewise, little is known about the relative importance of various parts of insulin’s covalent structure for the superstructural self-assembly. Here, by comparing the behavior of bovine insulin and its reduced and separated polypeptide chains under a variety of physicochemical aggregation regimes including agitation and the presence of TCEP, we attempt to elucidate these problems. Our results provide insight into the driving forces of the selfassembly of chiral superstructures of insulin amyloid.

The three disulfide bridges jointly impose a significant conformational restriction on the peptide, yet upon the formation of amyloid fibrils, the predominantly α-helical native structure converts into stacks of parallel β sheets with all the three disulfide bonds remaining intact.24 Because of the consecutive reduction in the number of accessible conformations, the persistent arrangement of S−S bonds within the amyloid fibril could be advantageous for developing structural models of insulin amyloid (as is the case in the work by Jimenez et al.15). The feasibility of the reduction of insulin disulfide bridges (partially25−27 or completely28−30) or the synthesis of non-native insulin isomers through S−S bond scrambling31 has allowed researchers to address questions about the relative propensities of A and B chains to form fibrils and the role of insulin’s conformational freedom in the selection of amyloidogenic pathways. The complete reduction of insulin releases free A and B chains whose unprotected sulfhydryl groups would normally be prone to fast reoxidation and formation of disulfide-linked oligomers in the presence of atmospheric oxygen. This can be circumvented either by the alkylation29 or oxidation of sulfhydryl groups to sulfonic acid.26,28,30 The main drawback of this approach lies in the fact that even tiny changes in the net electric charge20 and amino acid sequence32,33 of insulin affect its amyloidogenic properties, making the interpretation of the behavior of A and B peptides with protected −SH groups versus the whole insulin molecule more challenging. TCEP is a potent reducing agent34 that is suitable, unlike thiol compounds, for the reduction of protein disulfide bridges at low pH.35,36 This is advantageous for studies on insulin aggregation that typically takes place in an acidic environment: TCEP can act on insulin in situ during the formation of amyloid fibrils26 to protect reduced A and B chains from reoxidation. The self-assembly of amyloid superstructures with strong chiroptical properties was first observed for the agitationassisted aggregation of insulin.37 The process is abrupt, and its critical phase consists of the vortex-induced collapse of dispersed insulin amyloid into twisted superstructures of laterally aligned fibrils.14 In the absence of agitation and Debye screening of positively charged insulin particles provided by high ionic strength (from dissolved NaCl), insulin aggregation does occur although at a markedly decelerated rate. Several recent studies have shown that hydrodynamic forces (e.g., shear flow) not only trigger insulin aggregation38 but also dramatically affect the type of amyloid superstructure formed, for instance, by favoring the lateral association of fibrils over the formation of spherulites that takes place under quiescent conditions.39,40 Our earlier studies suggested that both agitation and the presence of salt are conditions sine qua non for the formation of chiral amyloid superstructures.12−14 However, Kurouski et al. have found recently that insulin amyloid fibrils grown in the absence of agitation may exhibit a −ICD signal after staining with ThT.41 The chirality of insulin chiral amyloid superstructures is not defined locally according to morphological and microscopically accessible traits (handedness of twisted aggregates) but on the macroscopic level by the sign of the extrinsic Cotton effect induced in thioflavin T (ThT), an amyloid-binding stain that is easily detected through induced circular dichroism (ICD) at 450 nm, approximately the wavelength of the ThT electronic transition. ThT is a molecular-rotor-type fluorophore with a pronounced affinity for amyloid surfaces.42 The gradual twisting of a free ThT molecule around the single C−C bond linking its benzothiazole

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MATERIALS AND METHODS

Insulin from bovine pancreas, TCEP, and deuterium chloride used for pD adjustment (35 wt % DCl solution in D2O, 99 atom % D) were from Sigma-Aldrich, US. D2O (“99.8 atom % D” grade) was from ARMAR Chemicals, Switzerland. Preparation of Reduced A and B Chains of Insulin. We have developed an effective procedure aimed at producing free reduced insulin A and B chains on a scale of tens of milligrams. It comprises simple step-gradient ion-exchange chromatography of reduced insulin followed by desalting of the peptides through solid-phase extraction and dialysis. The yields were 80% for the A chain and 60% for the B chain. As demonstrated by HPLC chromatography (Figure S1, Supporting Information), final peptide preparations have over a 95% degree of purity. The identity and the redox state of cysteine residues were confirmed by mass spectrometry. Obtained molecular mass values were 2340.0 and 3399.3 Da for the A and B chains, respectively (theoretical masses of reduced peptides being 2339.6 and 3399.9 Da for chains A and B, respectively). Procedure. The procedure of preparation of reduced A and B chains was as follows. A 30 mg portion of bovine insulin was dissolved in 2 mL of 100 mM glycine/NaOH buffer at pH 10.4 containing 52 mM DTT and 1 mM EDTA. The sample was incubated for 45 min at 60 °C and then diluted to 10 mL using 20 mM glycine/NaOH buffer at pH 10.4 containing 14% (w/v) NaCl, 1 mM DTT, and 0.5 mM EDTA. After cooling to room temperature, the sample was filtered through 0.45 μm filter and immediately subjected to ion-exchange chromatography on a MonoQ HR 5/5 column (LKB/Pharmacia, Sweden). The separation of A and B peptides was carried out on an LKB/Pharmacia FPLC system using a GP-250 Plus gradient programmer and two P-500 pumps. Two buffers were used: buffer I − 20 mM glycine/NaOH pH 10.4, 1 mM DTT, 0.5 mM EDTA; buffer II − 20 mM glycine/NaOH pH 10.4, 1 M NaCl, 1 mM DTT, 0.5 mM EDTA. The flow rate was set at 1 mL/min, and the spectrophotometric detection was carried out at 280 nm. The whole sample was injected onto the column equilibrated with 14% buffer II, and the void volume of the column containing the insulin B chain was collected for approximately 13 min. The column was then flushed for the next 5 5272

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detector. Typically, for a single spectrum 256 interferograms of 2 cm−1 resolution were coadded. During measurements, the sample chamber was continuously purged with dry CO2-depleted air. All spectra were corrected by subtracting the proper number of buffer and water vapor spectra prior to being baseline-corrected. Data processing was performed using GRAMS software (ThermoNicolet, US). Other details have been described previously.33

min, and after this the concentration of buffer II was set at 80% and the fraction containing the insulin A chain was collected for approximately 2.5 min. Both collected fractions were then acidified to pH 3.0 with HCl. To avoid problems with the low solubility of the A chain and by assuming a large volume of the fraction containing the B chain, we further processed both of these preparations separately. The fraction containing the insulin B chain was desalted and concentrated using a C-18 SepPak cartridge (Waters) equilibrated in 5 mM HCl. After thorough flushing with 5 mM HCl, the peptide was eluted from the column with 80% acetonitrile (v/v) containing 5 mM HCl. However, the fraction containing the A chain was dialyzed at room temperature against water acidified to pH 3.0 with HCl. The dialysis was carried out in 500−1000 MWCO Spectrapor CE dialysis tubing. Both desalted chains were then lyophilized and stored in a dry form at −20 °C. The degree of purity of the obtained peptide samples was assessed using HPLC. Chromatographic separation was carried out on a P680 HPLC system (Dionex, US) equipped with a Supelcosil LC-18-DB 4.6 mm × 250 mm column (Sigma, US) using two buffers: buffer III − 0.1% trifluoroacetic acid (TFA); buffer IV − 0.07% TFA, 80% acetonitrile (both v/v). The flow rate was maintained at 1 mL/min, and the spectrophotometric detection was carried out at 220 nm. The B-chain peptide was dissolved in buffer III, and the A chain was dissolved in buffer III supplemented with 6.7 M guanidine hydrochloride. The samples were injected onto the column equilibrated in 40% buffer IV, and the separation was carried out using the gradient 0.0−5.2 min, 40.0−48.5% IV; 5.2−13.9 min, 48.5−55.0% IV; 13.9− 14.0 min, 55−100% IV; 14.0−17.0 min, 100% IV; 17−17.2 min, 100− 40% IV; and 17−2−23.0 min, 40% IV. Fractions containing peptide chains were collected and lyophilized. For electrospray (ESI) mass spectrometry, peptide samples were redissolved in 0.1% formic acid plus 50% methanol (both v/v) and subjected to measurements using the HCT instrument (Bruker, Germany). Peptide Aggregation Procedure. Aggregates of insulin and its A and B peptides were obtained through either quiescent incubation or vortex mixing at 1400 rpm (using an Eppendorf thermomixer comfort accessory) of 0.25 wt % solutions of precursors in 0.1 M NaCl in D2O, pD 1.9 adjusted with diluted DCl (where pD is the pH-meter readout uncorrected for isotopic effects) at 60 °C. H2O was typically replaced with D2O because this facilitated in situ FT-IR measurements. Samples were incubated in airtight Eppendorf tubes in order to prevent reoxidation with atmospheric oxygen. To maintain reducing conditions, 20 mM TCEP was added before the onset of aggregation to selected samples (as specified in the figure captions). In the case of the coaggregation of A and B chains (data in Figure 6), the two reduced peptides were mixed in an equimolar ratio whereas their total concentration was maintained at 0.25 wt %. Depending on the regime of aggregation, freshly prepared samples of the amyloidogenic precursors were subjected to 4 days of vortexing at 1400 rpm or 7 days of quiescent incubation (in either case at 60 °C). Subsequently, insoluble aggregates were subjected to AFM/ICD/FT-IR analysis. AFM. Collected samples of aggregates were diluted 60-fold with deionized water. A small droplet (8 μL) of the fibril suspension was swiftly deposited onto freshly cleaved mica and left to dry overnight. AFM tapping-mode measurements were carried out using a Nanoscope III atomic force microscope from Veeco (US) and TAP300-Al sensors (resonance frequency 300 kHz) from BudgetSensors (Bulgaria). Other experimental parameters were the same as in earlier studies.14 ICD Spectroscopy. Insulin samples in acidified D2O-based solutions (as prepared for FT-IR measurements) were further diluted 20-fold with 0.1 M NaCl at pH 1.9 to the final aggregate concentration of 10−2 wt % whereas the molar concentration of added ThT was 45 μM. All ICD spectra were collected at 25 °C and under quiescent conditions on a J-815 S (Jasco, Japan) spectropolarimeter using 10 mm quartz cuvettes. FT-IR Spectroscopy. For FT-IR measurements, a CaF2 transmission cell equipped with a 0.025 mm Teflon spacer was used. All FT-IR spectra were collected on a Nexus FT-IR spectrometer (ThermoNicolet, US) equipped with a liquid-nitrogen-cooled MCT

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RESULTS AND DISCUSSION Infrared and ThT-ICD spectra of insulin amyloid fibrils grown in quiescent or agitated solutions and in the presence or absence of TCEP are shown in Figure 2. The shape and

Figure 2. (A) Amide I′ infrared band of amyloid fibrils formed upon incubation at 60 °C of acidified insulin under quiescent conditions (black) or agitation at 1400 rpm (red) in the presence (dotted lines) or absence (solid lines) of TCEP. (B) Corresponding ICD spectra of insulin fibrils obtained after staining with ThT.

position (at 1627 cm−1) of the conformation-sensitive amide I′ vibrational band of aggregates formed in the absence of both agitation and the reducing agent are typical for stacked parallel β sheets, the main constituent of insulin amyloid.15 We showed earlier that the agitation of aggregating insulin solutions not only accelerates the process but also leads to the formation of amyloid superstructures with distinct morphological and spectral properties.12 At 60 °C and 1400 rpm, ICD fibrils are formed, and this is reflected by the negative sign of the extrinsic Cotton effect induced in bound ThT (Figure 2B). The association of insulin protofilaments into chiral superstructures may affect the local twist of the β sheet and the strength of inter-β-strand hydrogen bonding, which are likely to underlie 5273

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transmission electron microscopy.26 This is in contrast to the well-defined twisted aggregates of −ICD fibrils shown in Figure 3C and described in our previous works.13,14 However, as the AFM image in Figure 3D proves, agitation induces the assembly of these superstructures only as long as insulin molecules retain an intact arrangement of disulfide bridges: the aggregates are clearly fibril-like but lacking the orderly lateral alignment visible in Figure 3C. Taken together, the AFM and FT-IR data suggest that native insulin or its early prefibrillar amyloidogenic intermediate states are prone to reduction by TCEP under the conditions of quiescent aggregation (i.e., when amyloid growth is relatively slow and the lifetime of forms of insulin that are vulnerable to TCEP is long). The aggregation of polypeptide molecules into insoluble precipitates effectively sequesters surviving S−S bonds from soluble TCEP. Agitation is well know to accelerate both phases of amyloidogenesis (i.e., nucleation (at the water−air interface) and elongation (by intensifying the migration of precursor molecules to fibril ends and multiplying the number of those ends by breaking down long fibrils)) and is therefore likely to facilitate the conversion of insulin to a TCEP-insensitive mature amyloid (Figure S2, Supporting Information). Still, the imperfect packing of aggregated fibrils seen in Figure 3D and their ICD-silent complex with ThT (Figure 2B) indicate that even sporadic disulfide-breakage events produce insulin molecules incapable of proper insertion into the −ICD scaffold without disrupting its morphological fabric and chiroptical properties. Previously published Raman data on TCEP-reduced insulin under quiescent conditions showed evidence of residual S−S bridges: the slow precipitation of “reduced” amorphous aggregates apparently prevented the completion of the reduction process.26 It is therefore unclear whether the formation of ICD-silent amorphous aggregates with the infrared features characteristic of the antiparallel β sheet is a property of the selfassembled equimolar mixture of A and B chains or rather a consequence of covalently inhomogeneous samples with the S−S bonds acting as topological traps. A comparative assessment of the propensities of free A and B peptides to form superstructures upon agitation was possible because of the highly efficient method of their separation in reduced forms from bovine insulin as described in this work (Materials and Methods). Using these peptides with free unprotected −SH groups (but in the presence of TCEP in order to prevent reoxidation) as amyloidogenic precursors allowed us to (i) avoid the aforementioned potentially problematic covalent modifications and (ii) gain insight into how conformation-trapping inter- and intrachain disulfide bonds affect the course of aggregation. The latter was achieved by comparing the aggregation of reduced A and B chains in the presence and absence of TCEP. In Figure 4, AFM images of aggregates of A (panels A−D) and B chain (panels E−H) formed under a variety of conditions are juxtaposed. Nonfibrillar ball-like aggregates with a diameter of roughly 20 nm precipitate from acidified solutions of A peptide in the absence of TCEP and agitation (Figure 4A). These amorphous entities contrast with fibril-like aggregates of A peptide with oxidized thiol groups described by Devlin et al.30 This and the fact that the reduced A-chain peptide does form thin amyloid protofilaments in the presence of TCEP (Figure 4B) strongly suggest that oxidation-triggered random disulfide bonds underlie the pronounced departure from fibrillar morphology. The two AFM images shown in panels C and D are typical of vortexed A-peptide aggregates without and with the addition of the

the subtle splitting of the corresponding amide I′ band (components at 1630 and ca. 1624 cm−1, Figure 2A).44 A more dramatic spectral change is evoked by the presence of TCEP during the relatively slow aggregation of nonagitated insulin: the amide I′ band becomes exciton-split into two peaks at 1615 (major) and 1684 (minor) cm−1, which are indicative of intramolecular antiparallel β sheets typical of amorphous aggregates. A similar observation was made by Zako et al., who studied insulin aggregation at 70 °C and without agitation.26 Interestingly, the coupling of the two perturbations (agitation and TCEP) restores the amyloid-like β sheet of aggregates according to the corresponding FT-IR spectrum (Figure 2A). Although the amide I′ band downshifts to the borderline region of the parallel/antiparallel β-sheet assignment (1619 cm−1), there is no evidence of the high-energy/low-intensity component at ca. 1684 cm−1, thereby suggesting that a parallel β sheet is formed.44 The similarity of the infrared features of vortex-induced aggregates in the presence and absence of TCEP is not echoed in the corresponding ICD spectra (Figure 2B). Amplitude AFM images collected for all four types of insulin aggregates are displayed in Figure 3. The tendency of

Figure 3. Amplitude AFM images of insulin aggregates obtained under different conditions. The preparation routine for all samples is the following: insulin dissolved at 0.25 wt % in 0.1 M NaCl, pD 1.9, aggregation at 60 °C. Variable conditions: quiescent aggregation in the absence (A) and presence of TCEP (B); agitation-assisted aggregation in the absence (C) and presence of TCEP (D). Insets show cross sections of selected representative specimens (according to the height images).

hydrophobic bovine insulin amyloid to agglomerate (especially in the presence of NaCl, as is the case in this study) is reflected in the majority of fibrils being piled together. Few remaining singly dispersed specimens enabled in situ AFM height measurements (the diameter of a representative fibril was ca. 6 nm, Figure 3A). The AFM image in the ensuing panel B confirms what was expected on the basis of the infrared data: insulin aggregates precipitating from nonagitated samples containing TCEP are highly amorphous. Few individual short fibrils protrude from clumps of porridgelike aggregates. Similarly, the formation of thinner curl-like insulin protofilaments was previously observed in the presence of TCEP using 5274

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Figure 4. Amplitude AFM images of aggregated insulin A chains (A−D) and B chains (E−H) under various regimes: in the absence of TCEP and agitation (A, E), in the presence of TCEP but under quiescent conditions (B, F), and upon agitation-assisted aggregation in the absence (C, G) and presence (D, H) of TCEP. The insets show cross sections of selected representative specimens (according to height images).

reducing agent, respectively. In either case, only amorphous aggregates are detected. Apart from the rather unsurprising outcome in the former case, the appearance of small clumps of associated particles in samples vortex mixed in the presence of TCEP implies that the protofilaments shown in Figure 4B are very brittle and as such disintegrate upon agitation into shorter stretches with smaller aspect ratios that are more likely to withstand hydrodynamic forces. This reminds us of the fragility of covalently intact insulin fibrils that self-assemble into spherulites without agitation and form nematic phases in an intermediate range of the agitation rate.40 AFM images of B-peptide aggregates formed under identical sets of conditions reveal an entirely different behavior from that of the A-chain peptide. Even without the addition of TCEP, the B-peptide forms regular, slightly bent protofilaments that are approximately 3 nm in diameter (Figure 4E), which demonstrates that it is markedly less prone to entanglement in disulfide bond networks than A chains. This can be rationalized by the presence of only half the number of cysteine residues relative to the A chain (two vs four), which are furthermore located in remote parts of the B chain (Figure 1). Whereas the thickness of B-chain fibrils assembled under quiescent conditions either in the absence or presence of TCEP (Figure 4F) compares well with the AFM data on aggregates of oxidized B chains, they are not as straight as observed in the study by Devlin et al.30 In fact, the tendency to bend was noted for amyloid fibrils composed of reduced unmodified B chains of insulin in the earlier work by Fink’s group.29 Hence, the oxidation of the B-peptide’s cysteine side chains seems to increase the persistence length of the fibrils. The most striking difference between the two peptides is that in the case of the B peptide, agitation leads not to amorphization but rather to the lateral self-assembly of fibrils into superstructures reminiscent of −ICD insulin aggregates (Figure 4G,H vs Figure 3C). The process is complete only in the presence of TCEP, again suggesting that even partial oxidation of the sulfhydryl groups decreases the capacity of individual B-peptide fibrils to merge into higher-order entities.

An immediate conclusion arising from the AFM data alone is that the properties and dynamics of the B chain are the driving force for the self-assembly of chiral superstructures of insulin amyloid. This is confirmed by the corresponding ThT-ICD spectra: out of eight types of samples analyzed in Figure 4, only the B peptide agitated with TCEP produced aggregates with a nonzero ICD signal (Figure 5).

Figure 5. ICD spectra of ThT-stained aggregates of the A and B chains obtained under the same conditions as in Figure 4. Only aggregates formed through the agitation of B-chain samples containing TCEP give a measurable ICD signal (a). The spectra for all other aggregates are flat with corresponding ICD signals below the limit of detection (b).

The intriguing case of vortex-mixed insulin, which under reducing conditions does not form chiral superstructures (Figures 2B and 3D), could be interpreted as a consequence of the exceedingly heterogeneous composition of the precursor solution (different types of partially reduced insulin molecules and free A and B chains). We have addressed this possibility by carrying out the same set of AFM/ThT-ICD experiments as in Figures 4 and 5 on an equimolar mixture of both peptides. In the AFM image showing the coaggregate of A and B chains obtained without agitation and TCEP, the characteristic shapes of aggregates of the A peptide (balls) and B peptide (fibrils) are visible (Figure 6A). Interestingly, all of the A-peptide clumps appear to sit on B-peptide fibrils, implying a degree of affinity 5275

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necessary long-range excitonic couplings that are likely to underlie the observed chiroptical properties of aggregates.45 In this context, the importance of the homogeneity of precursors and the singularity of amyloidogenic pathways becomes obvious. In conclusion, the reduced forms of both A and B chains of bovine insulin show marked tendencies to aggregate and form fibrils. Abundant with sulfhydryl groups, the A peptide is more vulnerable to entanglement in networks of disulfide bonds resulting in amorphous aggregation, whereas its fibrous aggregates formed under reducing conditions are deformed upon agitation. The B-chain peptide forms amyloid-like protofilaments even in the absence of TCEP; however, the reducing environment is necessary for the self-assembly of defect-free chiral superstructures of B-peptide chains. Our study highlights the sensitivity of fibrillar superstructures to local defects in the covalent structure of their building blocks and indicates the feasibility of using peptide fragments as amyloidogenic precursors for the synthesis of nanomaterials based on chiral amyloid superstructures.

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Figure 6. Amplitude AFM images of aggregates of equimolar mixtures of A and B chains formed under different conditions. The total concentration of both chains was 0.25 wt %. The peptides were dissolved in 0.1 M NaCl at pD 1.9. Aggregation was carried out at 60 °C. Variable conditions: quiescent aggregation in the absence (A) and presence of TCEP (B) and agitation-assisted aggregation in the absence (C) and presence of TCEP (D). Insets show cross sections of selected representative specimens (according to height images). The 1.5 μm scale bar is valid for all four panels.

ASSOCIATED CONTENT

S Supporting Information *

HPLC of separated reduced A and B chains. Additional FT-IR and ICD spectra of TCEP-treated insulin amyloid superstructures. Cross sections of AFM images. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +48 22 8220211 ext. 528. Fax: +48 22 822 5996. Email: [email protected].

between the two types of aggregates. Its specific character is unclear; although there is a significant difference in isoelectric point values (3.79 for A chain and 6.90 for B chain), both peptides have net positive charges in the extremely acidic environment of aggregation. In the presence of TCEP (panel B) or upon vortex mixing (C), aggregates with rather uniform morphologies resembling those of aggregates of the B peptide are formed (Figure 4F,G). However, under the conditions triggering the formation of ordered chiral superstructures of Bpeptide fibrils (TCEP and agitation), the mixture of A and B chains converts into stacks of fibrils similar in terms of their overall thickness (Figure S3, Supporting Information) but lacking the precise long-range ordering of the −ICD insulin aggregates. None of these four types of coaggregates showed a detectable ICD signal after being stained with ThT (data not shown). According to Figure 4, the presence of the reducing agent is necessary for the sufficient release of B chains from disulfide traps before the chiral superstructures could be assembled, but paradoxically, its addition to vortex-mixed insulin prevented the same process (Figure 3D). Hypothetically, the dynamics of B chains are the driving force for the assembly of the superstructures, but these may not be disturbed by the presence of foreign amyloidogenic peptides (partially reduced insulin or A chain), which would sway the amyloidogenic pathway of the B chain or would act as inclusion bodies in defect-ridden superassemblies. Although the shorter A chain exhibits no propensity to form flow-driven amyloid superstructures, it becomes apparently complicit as long as its own dynamics are controlled by the firm attachment to the B chain provided by the intact covalent structure of insulin. It has been argued that the defect-free alignment of fibrils is necessary not only to build ordered superstructures but also to provide

Present Address §

Collegium Medicum, Jagiellonian University, Sw. Anny 12, 31008 Cracow, Poland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Polish Ministry of Education and Science (grant NN 301 101236 to W.D.). We thank Ms. Oliwia Bochenska for mass spectrometry measurements. The preparation of reduced A and B chains was carried out with equipment obtained with the support of European Union structural funds (grants POIG.02.01.00-12-064/08 and POIG.02.01.00-12-167/08).

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ABBREVIATIONS AFM, atomic force microscopy; DTT, dithiothreitol; FT-IR, Fourier transform infrared; ICD, induced circular dichroism; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; ThT, thioflavin T

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REFERENCES

(1) Chiti, F.; Dobson, C. M. Protein Misfolding, Functional Amyloid, and Human Disease. Annu. Rev. Biochem. 2006, 75, 333−366. (2) Fowler, D. M.; Koulov, A. V.; Balch, W. E.; Kelly, J. W. Functional Amyloid - from Bacteria to Humans. Trends Biochem. Sci. 2007, 32, 217−224. (3) Scheibel, T.; Parthasarathy, R.; Sawicki, G.; Lin, X. M.; Jaeger, H.; Lindquist, S. L. Conducting Nanowires Built by Controlled SelfAssembly of Amyloid Fibers and Selective Metal Deposition. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4527−4532.

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