Complexation of Amyloid Fibrils with Charged Conjugated Polymers

Mar 11, 2014 - Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India .... Scientific Reports 2016 6...
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Complexation of Amyloid Fibrils with Charged Conjugated Polymers Dhiman Ghosh,† Paulami Dutta,† Chanchal Chakraborty,§ Pradeep K. Singh,† A. Anoop,† Narendra Nath Jha,† Reeba S. Jacob,† Mrityunjoy Mondal,† Shruti Mankar,† Subhadeep Das,†,‡ Sudip Malik,§ and Samir K. Maji*,† †

Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, Maharashtra, Mumbai 400076, India ‡ IITB-Monash Research Academy, Indian Institute of Technology Bombay, Mumbai, Maharashtra, Mumbai 400076, India § Polymer Science Unit, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 700032, India S Supporting Information *

ABSTRACT: It has been suggested that conjugated charged polymers are amyloid imaging agents and promising therapeutic candidates for neurological disorders. However, very less is known about their efficacy in modulating the amyloid aggregation pathway. Here, we studied the modulation of Parkinson’s disease associated α-synuclein (AS) amyloid assembly kinetics using conjugated polyfluorene polymers (PF, cationic; PFS, anionic). We also explored the complexation of these charged polymers with the various AS aggregated species including amyloid fibrils and oligomers using multidisciplinary biophysical techniques. Our data suggests that both polymers irrespective of their different charges in the side chains increase the fibrilization kinetics of AS and also remarkably change the morphology of the resultant amyloid fibrils. Both polymers were incorporated/aligned onto the AS amyloid fibrils as evident from electron microscopy (EM) and atomic force microscopy (AFM), and the resultant complexes were structurally distinct from their pristine form of both polymers and AS supported by FTIR study. Additionally, we observed that the mechanism of interactions between the polymers with different species of AS aggregates were markedly different.

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INTRODUCTION Amyloid fibrils are highly ordered protein/peptide aggregates that have been associated with many human diseases including Alzheimer’s and Parkinson’s disease.1,2 Amyloid formation is a nucleation-dependent polymerization process3,4 in which native proteins are converted into aggregation-prone “partially folded intermediates”5,6 that subsequently self-assemble into oligomers (nucleus), eventually forming mature fibrils. It was hypothesized that amyloid formation is a generic property of polypeptide chains7 because many proteins/peptides form amyloid under certain conditions.8,9 Proteins with different primary and secondary structures form amyloid with common characteristics such as the cross-β-sheet structure,1,2,10 which allow proteins to bind to dyes such as Thioflavin T (ThT)11 and Congo Red (CR).12 Recent studies have suggested that amyloid fibrils are not the primary toxic species responsible for human diseases; rather, they may serve as nontoxic scavengers of toxic oligomers.13−15 The major pathological hallmarks of Parkinson’s disease (PD) is the presence of insoluble, fibrous aggregates composed of 140 residue protein, α-synuclein (AS) in intraneuronal inclusions of Lewy bodies (LBs), and Lewy neuritis (LNs).16 The animal models including transgenic fly and others of © 2014 American Chemical Society

synucleinopathies recapitulate many features of the human disease, including LBs-like fibrillar AS inclusions, loss of dopaminergic neurons, and motor abnormalities, which support direct involvement of AS aggregation in PD pathogenesis.17,18 AS is a highly conserved protein that is predominantly expressed in neurons, particularly in the presynaptic terminals,19 and may have a role in synaptic plasticity.16,20,21 The AS is soluble in water or in physiological buffer due to its high net charge and has been shown to be a natively unstructured conformation.16,20−23 The AS consists of an N-terminal region (residues 1−60) with a highly conserved hexamer motif KTKEGV, reported to form α-helices in association with membranes.24,25 The central hydrophobic region containing “non-amyloid β component” (NAC) (residues 61−95) is shown to be the key responsible amino acid region for aggregation.26,27 The C-terminal region is however (residues 96−140) highly acidic but has been shown to regulate AS aggregation.28 Received: May 28, 2013 Revised: February 9, 2014 Published: March 11, 2014 3775

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effectively interacted with monomers. The data collectively indicate that complexion of AS oligomers and fibrils with polymers is one of the possible reasons for the acceleration of AS fibril formation.

Many biological polymers such as glycosaminoglycans are known to interact with amyloidogenic protein including AS and modulate their aggregation.22,29−32 These polymers are also known to bind to preformed amyloid fibrils and thereby increase their stability against degradation. Interestingly, not only biological polymers but also synthetic conjugated polymers are known to bind to preformed amyloids in vitro and in vivo and therefore raise the possibility for application as amyloid imaging agents.33−35 Moreover, it has been shown that conjugated polymers could be able to differentiate the different prion strains36 and different conformational states of Aβ fibrils,33 associated with Alzheimer’s disease (AD). The conjugated polymers are also known inhibit the prion propagation.37 Moreover, several other elegant studies have also shown that conjugated polyelectrolytes and/or polymers make complexes with amyloid fibrils, which can be used for synthesis of new functional materials.38,39 Although many previous studies were performed to establish the interaction/ complexation of amyloid and conjugated polymers, how these conjugated polymers modulate the amyloid aggregation is largely unknown. Additionally, the role of charged side chains of these polymers in amyloid complexation has also not been systematically studied yet. Here, we studied the mechanism of AS aggregation in the presence of two oppositely charged polyfluorene polymers. Polyfluorene polymers are known for their usefulness in biological/chemical sensing.40,41 The aggregation and conformational transition of AS in the presence and absence of two oppositely charged, water-soluble conjugated polyfluorenes (PF and PFS) (Figure 1) was performed using circular dichroism

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EXPERIMENTAL SECTION

Chemicals and Reagents. All chemicals used were of highest purity grade and purchased from SIGMA, USA. Water was double distilled using a Milli Q water purification system. Synthesis of PF and PFS. Polyelectrolyte polymer PF was synthesized by polymerizing functionalized 2,7-dibromo-9,9′-bis(6″bromohexyl)fluorene with 1,4-phenyldiboronic acid by the Suzuki cross-coupling procedure and thereafter by quarternization using triethylamine (TEA) as per our previously reported procedure.42 PFS was synthesized by functionalized 2,7-dibromo-9,9′-bis(6″bromohexyl)fluorene with 1,4-phenyldiboronic acid by Suzuki coupling, and then a postpolymer reaction was performed with phenol, concentrated sulfuric acid, and subsequent stepwise addition of NaOH.40 The molecular weight (Mw) of polymer poly(9,9′-bis(6″bromohexyl)fluorene-co-alt-1,4-phenylene), which was synthesized after Suzuki polymerization, is ∼8000 Da, and the polydispersity index (PDI) is 1.75, determined by gel permeation chromatography (GPC) using tetrahydrofuran (THF) as the eluent and polystyrene as a standard. PF and PFS both were sparingly soluble in water, and maximum solubility was obtained around 25 μM. To increase the concentration of these polymers, a THF−water mixture was used. In 5% THF, the solubility of these polymers was increased drastically and solubility up to 2.5 mM was achieved. Stock solutions of these polymers were stored at room temoerature for further study. Protein Purification. AS was expressed in Escherichia coli BL21 (DE3) strain, and protein was purified according to the published protocol by Volles et al.43 with slight modification.44,45 Briefly, after expression, bacterial cells were pelleted down by centrifugation and the pellet was resuspended in buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 150 mM NaCl) with protease inhibitor cocktail (Roche, USA) and sonicated. The sonicated cell suspension was heated for 20 min in boiling water and then centrifuged (14 000g, 30 min). Supernatant was collected, and 10% streptomycin sulfate (136 μL/ml supernatant) and glacial acetic acid (228 μL/ml supernatant) were added to the supernatant followed by centrifugation (14 000g) for 30 min to revome the DNA. Supernatant was removed, and protein was precipitated using an equal volume of saturated ammonium sulfate (4 °C) solution. Precipitated protein was further washed with ammonium sulfate solution. The washed pellet was resuspended in 100 mM ammonium acetate (4 °C) and again precipitated by an equal volume of absolute ethanol. Ethanol precipitation was repeated twice. Protein was resuspended in 100 mM ammonium acetate, lyophilized, and stored at −20 °C until further use. Purity of the proteins was confirmed by SDS-PAGE. MALDI-TOF mass spectrometry was used to further validate the molecular mass of the protein, which showed m/z of 14.4 Kda. Preparation of Low Molecular Weight (LMW). To prepare the seed-free solution of AS, solid lyophilized AS was dissolved in PBS buffer, pH 7.4, 0.01% sodium azide. AS is a 140 amino acid residue protein, and its isoelectric point (pI) is 4.67. It contains 24 acidic amino acid residues and 15 basic amino acid residues. Most of the acidic amino acid residues are located at the C terminus.46 This makes the C terminus of the protein negatively charged and also gives a net negative charge to the AS protein at pH 7.4. Solid AS is not fully soluble in buffer, pH 7.4, as reported earlier.45 A few microliters of 2 M NaOH was added to dissolve the protein, and then the pH was adjusted to 7.4 by adding few microliters of 2 M HCl. This solution was then centrifuged at 4 °C for 30 min at 14 000g to remove any preexisting fibrillar aggregates. Then the AS solution was kept for overnight dialysis (10 kDa membrane cut off) using PBS, pH 7.4, 0.01% sodium azide. After 12 h of dialysis, the solution was added to 100 kDa cut-off filters (YM-100) and centrifuged for 30 min at 4 °C. The supernatant flow-through was collected. This preperation of AS mostly contain monomer along with a low amount of oligomers.44

Figure 1. Chemical structure of polymers and amino acid sequence of AS. Chemical structure of PF and PFS (top) showing their backbone and side-chain structures. Amino acid sequence of AS (bottom) with one-letter codes showing the 140 amino acid residues.

(CD) spectroscopy and Thioflavin T (ThT) fluorescence. In addition, incorporation of the polymers into the AS fibrils was studied morphologically using atomic force microscopy (AFM) and transmission electron microscopy (TEM) and structurally using Fourier transform infrared spectroscopy (FTIR). Both PF and PFS accelerated the AS aggregation and were incorporated within the network of amyloid fibrils. The morphologies of the complexes were different from those of either the AS amyloid or the polymers alone. The effects of AS on polymer properties were also studied, and the modified polymers were compared to their pristine forms. The size exclusion chromatography (SEC) profile of AS in the presence and absence of these polymers and the binding data suggested that PF and PFS interacted more preferably with fibrils and oligomers possibly due to the more hydrophobic interaction between AS fibrils/ oligomers and polymers. However, both polymers less 3776

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Previously, many studies have been performed using a similar approach for preparing soluble protein/peptide for fibrillation studies.15,44,47,48 We therefore name this species low molecular weight (LMW). The concentration of LMW was measured by a UV spectrophotometer as described before.45 AS Aggregation. To analyze how polymers of PF and PFS influence the AS aggregation, an aggregation study was performed with 150 μM protein concentration in the presence and absence of an equimolar concentration of both PF and PFS. Since both 2.5 mM PF and PFS stock solutions were made in 5% THF−water mixture, the resulting THF in the final solution became 0.3% (v/v). After this dilution, we did not observe any turbidity/precipitation, suggesting that 150 μM polymers were completely soluble in buffer containing 0.3% THF. Solutions were incubated in Eppendorf tubes (Eppendorf AG, Hamburg, Germany) for fibrillization at 37 °C with slight rotation (50 rpm). Only AS was also incubated in the presence of 0.3% (v/v) THF. During incubation, CD, polymer fluorescence, and ThT binding studies were performed at regular time intervals. Size Exclusion Chromatography (SEC). To study the interactions of PF and PFS with monomers and preformed AS oligomers, a SEC study was performed with soluble AS both in the presence and in the absence of polymers. For this, solid AS was dissolved in PBS, pH 7.4, at a concentration of 20 mg/mL. As described before, the AS was not fully soluble. We therefore added few microliters of 2 M NaOH to dissolve the protein, and the pH was adjusted to 7.4 by adding a few microliters of 2 M HCl. For polymer binding study, the solubilized AS solution was aliquoted into three different Eppendorf tubes. PF and PFS were added separately to two different tubes such that the final concentration of polymers became 150 μM. In a third tube (control), buffer was added to adjust the final volume as in the other tubes. The final concentration of AS in each tube was identical. The mixtures were then incubated for 30 min at room temperature and centrifuged at 14 000g for 30 min. The supernatant, 500 μL, was collected and injected to a SEC column. A 500 μL amount of AS, PF, and PFS alone was also injected to a SEC column as a control. The monomeric and oligomeric fractions were collected and immediately used for further study. The SEC elution peaks were integrated using Unicorn 5.2 (GE Healthcare) software. The area under each peak was calculated and analyzed. The exact concentration of monomeric and oligomeric fractions could not be measured at this point as the amount of polymers bound to these species is unknown. Therefore, for fluorescence and CD studies, an equal volume of sample was employed without normalizing its concentration. Circular Dichroism (CD) Spectroscopy. CD spectroscopy is a very helpful technique to determine the secondary structural transition of protein/peptides.49 To study the conformational changes of AS during aggregation in the presence and absence of polymers, a CD study was performed at regular intervals during incubation. To do this study, protein aliquotes were diluted to a 10 μM final concentration in 150 μL volume. Samples were placed into a 0.1 cm path-length quartz cell (Hellma, Forest Hills, NY), and CD spectra were acquired using a JASCO spectropolarimeter (model J-810). All measurements were done at 25 °C. Spectra were acquired in the wavelength range of 200− 260 nm. Three independent experiments were performed with each sample. Raw data were processed by smoothing and subtraction of buffer spectra. CD results were represented in molar ellipticity (K deg cm2 dmol−1). Thioflavin T (ThT) Binding. ThT is useful dye to study the time course of protein aggregation and amyloid formation.31,44 To perform the ThT binding study, 5 μL of incubated sample in PBS, pH 7.4, 0.01% of sodium azide, was diluted into 150 μL in the same buffer to a final concentration of 5 μM. The protein solution was then mixed with 2 μL of 1 mM ThT prepared in the same buffer, and fluorescence was measured immediately. Fluorescence measurement was done using Horiba-Jobin Yvon (Fluoromax 4) with excitation at 450 nm and emission in the range of 460−500 nm. Both the excitation and the emission slit widths of 5 nm were used for all studies. ThT fluorescence intensities at 480 nm were plotted against different incubation times, and data were fitted in a sigmoidal curve. The lag time was calculated according to the published protocol50 using

y = y0 + (ymax − y0 )/(1 + e−(k(t − t1/2))) where y is the ThT fluorescence at a particular time point, ymax is the maximum ThT fluorescence, y0 is the ThT fluorescence at t0, and tlag was defined as t lag = t1/2− 2/k. AS aggregation kinetics is a nucleation-dependent polymerization reaction, which can be explained by a two-step mechanism. In this mechanism, the first step is the nucleation phase, where monomer converted to the nucleus, and second step is the elongation phase, where nucleus binds to monomer to form fibrils. We analyzed the kinetics data of protein aggregation using a Finke−Watzky (F−W) two-step model consisting of nucleation (A → B) and autocatalytic growth (A + B → 2B) phases as shown in Scheme 1.51Here, k1 and k2

Scheme 1

are the rate of nucleation and rate of growth phase, respectively. ThT fluorescence is plotted against time, and rate constants are calculated by fitting to the equation

1+

k1 k 2[A]0 exp(k1 + k 2[A]0 )t

[A]0 is the initial concentration of the protein. Fourier Transform Infrared Spectroscopy (FTIR). FTIR is routinely used for determination of different secondary structural components of proteins/peptides.52,53 It is a particularly very useful technique for determining the β-sheet conformation in peptides/ proteins. For FTIR study, 8 days old samples of AS fibrils formed in the presence and absence of PF and PFS were used. In brief, an aliquot of 5 μL of each sample was spotted on a KBr pellet and dried immediately under an IR lamp, and then the spectra were acquired as an average spectrum of 32 scans using a Vertex-80 FTIR system (Bruker, Germnay). In order to analyze the secondary structural components of AS aggregated in the presence and absence of PF and PFS, FTIR spectra (1600−1700 cm−1 corresponding to amide-I region) were subjected to Fourier self deconvolution (FSD) followed by Lorentzian curve fitting. This drying method of FTIR study did not effect the secondary structure as our independent study with bovine serum albumin (BSA) and AS monomer showed a mostly helical and random coil conformation, respectively, as expected (data not shown). Atomic Force Microscopy (AFM). To analyze the morphology of AS fibrils formed in the presence and absence of PF and PFS, 150 μM aggregated samples were diluted to 15 μM concentration in PBS buffer, pH 7.4, 0.01% sodium azide, and spotted on a freshly cleaved mica sheet followed by washing with water. The mica was dried under vacuum desiccators. Imaging was done in tapping mode underetched silicon AFM cantilever using Veeco Nanoscope IV multimode AFM. At least five different areas of two independent samples were scanned with a scan rate of 1.0 Hz. Electron microscopy (EM). To furher analyze the morphology of protein aggregates, EM study was also perfromed with AS sample. For this, a protein aliquot was diluted in distilled water to a final concentration of ∼50 μM, spotted on a glow-discharged, carboncoated Formvar grid (Electron Microscopy Sciences, Fort Washington, PA), incubated for 5 min, washed with distilled water, and then stained with 1% (w/v) aqueous uranyl formate solution. Uranyl formate solution was freshly prepared and filtered through 0.22 μm sterile syringe filters (Millipore, USA). EM analysis was performed using a FEI Tecnai G2 12 electron microscope at 120 kV with nominal magnifications of 43 000× and 60 000×. Images were recorded digitally using the SIS Megaview III imaging system. At least two independent experiments were carried out for each sample. UV−Visible Spectroscopy. To analyze the interaction of AS and polymers, UV−visible spectroscopy was performed. To do this, a 5 μL aliquot of protein samples (150 μM) in the presence and absence of both polymers was diluted to 150 μL in PBS (pH 7.4) containing 3777

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0.01% (w/v) sodium azide and measured UV in the range of 300−500 nm using a JASCO UV−vis spectrophotometer (model V-650). The PF and PFS alone samples were also used as controls. Fluorescence Spectroscopy. Both PF and PFS are conjugated polymers, and they produce fluorescence when excited at their excitation maxima. Using the fluorescence property of PF and PFS, we determine the interaction of PF and PFS with different AS species. To do this, a 5 μL aliquot of each AS sample in the presence and absence of PF and PFS was diluted to 150 μL in PBS (pH 7.4) containing 0.01% (w/v) sodium azide. The PF and PFS fluorescence were performed with excitation at 370 and 340 nm, respectively, and emission in the range of 380−470 and 370−480 nm, respectively, with both excitation and emission slit widths of 5 nm. For Tyr fluorescence study, samples were excited at 280 nm and emissions were recorded in the range of 290−500 nm. All fluorescence spectra were measured using a Horiba-Jobin Yvon (Fluoromax 4) spectrofluorimeter. The monomeric AS proteins were collected from SEC and diluted three times before fluorescence measurement. Determination of Dissociation Constant (Kd). Using the increase in fluorescence intensity when both polymers bind to AS species, dissociation constants were determined for both polymers. For this, the PF and PFS concentration was kept constant at 2 μM and protein concentrations were varied. Different species of AS (monomer and oligomers), which were isolated from SEC, and preformed amyloid fibrils were added to the polymer solution with an increasing concentration from 0 to 30 μM. All samples were incubated for 1 h at room temperature. PF/PFS fluorescence was measured as described in the Fluorescence Spectroscopy section. PF produce emission spectra with two distinct peaks, one at 418 nm and another at 440 nm, whereas PFS produced a maximum fluorescence peak only at 418 nm. Fluorescence values at 418 for both PF and PFS were plotted against increasing concentrations of different AS species, and dissociation constants were determined using the software of Graph Pad. All fluorescence spectra were measured using a Horiba-Jobin Yvon (Fluoromax 4) spectrofluorimeter. PF and PFS Fluorescence Study in the Presence of NaCl. A 75 μM concentration of different AS species (monomer, oligomer, and fibrils) was taken in Eppendorf tubes, and 5 M NaCl solution in PBS buffer, pH 7.4, was added to it such that the final concentration of NaCl becomes 500 mM and AS concentration becomes 50 μM. Solutions were then incubated for 30 min. Next, PF and PFS solutions were added to each solution separately. In the final mixture, PF/PFS concentration becomes 50 μM. Solutions were again incubated for another 30 min. Afterward, monomer, oligomers, and fibrils bound PF and PFS fluorescence was recorded as described earlier with 4 μM protein/polymer concentration. Only 50 μM PF and PFS was incubated in the presence of 500 mM NaCl. PF/PFS fluorescence in the presence of NaCl was subtracted for each case and normalized to compare the effect of NaCl on the fluorescence intensity of polymers bound with monomers, oligomers, and fibrils. This enables us to measure the extent of electrostatic interaction for the binding of PF/ PFS with different species of AS. MTT Metabolic Assay. To analyze whether PF and PFS influenced the toxicity of AS fibrils, the 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assay54,55 was carried out. For this study, 150 μM AS protein was incubated for aggregation in the presence and absence of 150 μM each polymers as described earlier. To test the toxicity of these aggregates, neuronal cells of SHSY5Y were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Himedia, India) supplemented with 10% FBS (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin in a 5% CO2 humidified environment at 37 °C. Cells were seeded at a density of 10 000 cells per well on 96-well plates in 100 μL medium. After 24 h of incubation, the old culture media was replaced with fresh media containing 5 μM test samples (AS fibrils formed in the presence and absence of polymers, each of the polymers alone, buffer alone), and cells were incubated for another 36 h at 37 °C. After incubation, 10 μL of 5 mg/mL MTT (prepared in PBS) was added to each well and the incubation was continued for 4 h. Finally, 100 μL of a solution containing 50% dimethylformamide and 20% SDS (pH 4.7) was added

to each well and incubated. After overnight incubation in a 5% CO2 humidified environment at 37 °C, absorption values at 560 nm were determined with an automatic microtiter plate reader (Thermo Fisher Scientific, USA). Statistical Analysis. The statistical significance was determined by one-way ANOVA followed by Newman−Keuls multiple comparison posthoc test; *P < 0.05; **P < 0.01; NS P > 0.05.

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RESULTS AND DISCUSSION PF and PFS Accelerate AS Aggregation. The effects of PF and PFS (Figure 2) on AS aggregation were studied. The

Figure 2. PF and PFS accelerate AS aggregation. (A) CD spectroscopy of AS in the presence and absence of PF and PFS during aggregation showing acceleration of conformational transition during incubation. Selected CD spectra were shown for clarity. (B) ThT binding of AS, AS+PF, and AS+PFS during aggregation. ThT results showing the increase rate of AS fibrillation in the presence of both polymers. ThT fluorescence intensities were normalized for producing the plot.

polymer PF is cationic because of the ammonium moiety in its side chain, whereas PFS is anionic because of the presence of a sulfonate functional group. These ionic groups on the side chain of polymers also make them water soluble to some extent. The LMW15,45 form of AS was mixed with each of the polymers separately in a ratio of 1:1 (150 μM each), and the mixtures were incubated at 37 °C with slight agitations. AS and each of the polymers were also incubated separately as controls. CD spectroscopy and ThT binding were performed at regular intervals during incubation to monitor conformational transition and amyloid formation, respectively (Figure 2). Immediately after mixing with PF or PFS, LMW AS showed mostly random coil (RC) structure with single minima at ∼198 nm (Figure 2A, 0 h) in CD. Continuous monitoring of CD spectra showed that at 50 h, AS+PF displayed single minima at ∼218 nm, suggesting β-sheet conformation. AS+PFS also showed a conformational transition of RC → β-sheet at 75 h. AS alone did not show any major conformational transition and remained mostly as RC at this time point (Figure 2A). AS took almost 90 h to form a β-sheet structure. These results indicate that both PF and PFS accelerate the RC → β-sheet conformational transition of AS, irrespective of their side chain charges. ThT binding studies were also performed to monitor AS amyloid formation in the presence and absence of both polymers. ThT is a dye that binds to β-sheet-rich amyloids but not to monomeric proteins/peptides.11 ThT binding to 3778

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Figure 3. FTIR spectra of AS+polymer fibrils. FTIR spectra (1600−1700 cm−1 region) of AS fibrils (left) and AS fibrils formed in the presence of PF (middle) and PFS (right). Fibrils of AS−polymer complex showed significant differences in spectra from the AS fibrils alone.

amyloids results in a large increase in fluorescence at 480 nm when excited at 450 nm. AS aggregation is a nucleationdependent polymerization process,3 where at the beginning of aggregation significant ThT fluorescence is not observed. This time is most often referred as lag time. After the lag time, a high increase of ThT fluorescence is generally observed (fibril growth) until it reaches fluorescence saturation (stationary phase). Our ThT fluorescence data monitoring the kinetics of aggregation showed that at 0 h all samples produce insignificant ThT fluorescence (Figure 2B). However, an increase in ThT fluorescence occurs with progression of time. ThT of AS alone started increasing after 60 h and became stationary after 100 h. AS in the presence of PF showed faster aggregation kinetics. ThT binding of AS in the presence of PF was low up to 25 h and afterward started increasing until it saturated after 60 h. AS in the presence of PFS showed intermediate kinetics where an increase of ThT fluorescence was observed only after 50 h and ThT fluorescence reached saturation at 80 h. From these ThT binding curves, lag time was calculated using the previously published protocol,50 and lag time was found to be 25 ± 4.5, 45 ± 5, and 59 ± 6.5 h for AS+PF, AS+PFS, and AS, respectively. Rate constants were calculated from the ThT binding curve with the help of the previously described method.51 The nucleation rate for AS in the presence of PF was found to be (1.923 ± 0.11) × 10−4 h−1, and the elongation rate was (1.18 ± 0.09) × 10−3 μM−1 h−1. For AS in the presence of PFS, nucleation and elongation rates were found to be (1.719 ± 0.08) × 10−4 h−1 and (9.192 ± 1.2) × 10−4 μM−1 h−1, respectively. In the absence of any polymers, the rate of AS aggregation was found to be slowest. Nucleation and elongation rate constants were obtained for AS alone was of (10.269 ± 1.27) × 10−6 h−1 and (6.898 ± 1.5) × 10−4 μM−1 h−1, respectively. The ThT binding data therefore clearly suggests that both PF and PFS accelerate AS fibrillation significantly where the effect of PF is more prominent compared to PFS. As reported previously that heparin29 (negatively charged polysaccharide) and polyamines56 (positively charged polymers) accelerated AS aggregation, it is quite possible that irrespective of side chain charges both PF and PFS may bind to soluble AS species and increase their conversion to fibrils. The incubated polymer sample did not show any ThT fluorescence at 480 nm, suggesting that the polymer itself did not bind to ThT; rather amyloid that complexed with polymers binds the ThT and increases the ThT fluorescence. To study the competitive binding of PF/PFS and ThT to the amyloid, we also determined the ThT fluorescence of two different preparations. For one preparation, preformed fibrils were incubated with each of PF and PFS and then ThT solution was added. For another preparation, the preformed fibrils were first

incubated with ThT and then PF and PFS were added separately. The ThT fluorescence of these two types of sample for each polymer did not show any difference (Figure S1, Supporting Information), suggesting that polymers and ThT do not compete for binding to AS fibrils. Therefore, the extent of ThT fluorescence obtained in this study was purely from amyloid species that complexed with each of the polymers. Structural Characteristics of Amyloid−Polymer Complex. Structural characterization of AS fibrils formed in the presence and absence of PF and PFS was further characterized by FTIR spectroscopy. FTIR spectra were recorded from 1600 to 1800 cm−1 for all samples, and peaks were assigned in accordance with published reports.52,53 FTIR spectra (detail FTIR assignments are shown in Table S1, Supporting Information) data in the 1600−1700 cm−1 region, which is characteristic of protein secondary structures,52,53 are presented in Figure 3. The AS fibrils showed the most intense peaks at 1631 and 1620 cm−1, corresponding to the β-sheet structure, and the less intense peaks at 1669 and 1678 cm−1 , corresponding to the 310 helix and/or β-turn structure.53 Both the AS+PF and the AS+PFS fibrils showed a highintensity peak at ∼1631 cm−1 corresponding to the β-sheet structure. Interestingly, peaks corresponding to 310 helix and/or a β-turn showed a decrease in intensity and the random coil peak disappeared (Figure 3). PF or PFS did not show any peak in this region (Figure S2, Supporting Information). Detection of β-sheet character in both complexes indicates that after complex formation the amyloid structure was retained; however, one might assume a different structural organization than that in pure AS fibrils. Morphological Analysis of Fibrils. Two different techniques (AFM and EM) were used to analyze the morphological characteristics of the amyloid fibrils that formed in the presence and absence of both polymers (Figures 4). AS samples in AFM showed mostly elongated fibrils of few micrometers in length, although some small filaments were also observed (Figure 4A). The AS+PF image in Figure 4A shows thin filaments that are randomly embellished with each other and with the polymer agglomerates. The AS+PFS samples also produced short and clumped filaments (Figure 4A, AS+PFS image), and a few large PFS clusters were present within the fibril network. An apparent difference in the diameter of AS+PF and AS+PFS fibril samples compared to AS fibrils alone could be due to PF or PFS incorporation into the fibril structure in a manner that might alter the lateral association of individual filaments. EM was also used to visualize the morphology of AS fibrils formed in the presence and absence of the polymers. The incubated AS sample showed predominantly straight, un3779

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Figure 5. UV absorbance and fluorescence spectroscopy of AS− polymer complex. UV absorbance spectroscopy of AS in the presence and absence of PF and PFS for soluble LMW form (A) and fibrils (B) species. Time-dependent increase in PF (C) and PFS (D) fluorescence during AS fibrillation. An increase in absorbance and fluorescence were observed due to AS−polymer interactions.

Figure 4. Morphology of fibrils. (A) AFM images of fibrils formed in the presence and absence of PF and PFS; height scale on the top and amplitude scale on the bottom. (B) EM of AS fibrils in the presence and absence of PF and PFS. Fibrils formed in the presence of polymers showed significant differences in morphology compared to AS fibrils alone. Scale bars: 200 nm for EM and 500 nm for AFM images.

PF and PFS in the presence of the LMW AS showed a slight increase in absorption without any major wavelength maxima (λmax) shift (Figure 5A). Similar UV absorption profiles were also observed for the fibrils that formed in the presence of polymers where a significant increase in absorption (Figure 5B) was observed. The data suggest that the AS does not drastically affect the major spectral properties of the two polymers but increases the intensity of UV absorption. This enhancement of UV absorption intensity for both polymers in the presence of AS is possibly due to stronger interaction between polymers and AS species, and it eases π → π* transition of the conjugated segment of PF and PFS. As both PF and PFS are fluorescent polymers, their interaction with AS might affect their fluorescence properties. To determine the polymer fluorescence during AS aggregation, PF and PFS were incubated in the presence of LMW AS until fibril formation. During incubation, PF and PFS fluorescence were measured at regular intervals with excitation at 340 nm and emission in the 370−480 nm range for PFS and excitation at 370 nm and emission in the range of 380−470 nm for PF. The fluorescence intensities of AS alone and each of the polymers were also determined as controls. PF alone produced two intense fluorescence peaks at ∼420 and ∼440 nm (Figure 5C), whereas PFS produced only one peak at ∼420 nm (Figure 5D). During incubation at 37 °C with agitation, these fluorescence properties of pure polymers were mostly unchanged (data not shown). Interestingly, the AS+PF mixture at 0 h showed a small increase in fluorescence intensity compared to PF alone. During incubation, PF fluorescence was found to increase significantly without changing the λmax (Figure 5C), consistent with UV−vis spectroscopy. A similar trend was also observed for AS in the presence of PFS during incubation (Figure 5D). The increase in the fluorescence intensity of PF and PFS in the presence of AS aggregates might be due to inhibition of interchain interaction between polymer chains in AS+PF or AS +PFS complex, which delays the transition from the excited

branched fibrils of varying lengths, with fibril width ranging between 20 and 23 nm (Figure 4B). Polymer PF and PFS samples were mostly of large agglomerate, with the latter showing more clumped globules (Figure S3, Supporting Information). Representative EM images of AS+PF complex (Figure 4B) showed that the fibrils were mostly decorated with PF polymers and appear as small globular structures aligned on the fibrils. These fibrils showed increased lateral association of filaments, resulting in an average fibril width of 27 nm. In the AS+PFS sample, PFS appeared to be deposited or entrapped in the AS fibril networks (Figure 4B, AS+PFS). In this case, only thin filaments were observed, with less lateral associations (average fibril width ≈ 17 nm). The different fibril morphology suggests that the lateral associations of filaments in AS fibrils were significantly affected in the presence of PFS compared to PF. Moreover, only two filaments were associated to form mature fibrils in the case of AS+PFS as opposed to >3 filaments for AS or AS+PF samples. The EM results therefore confirm that AS in the presence of these two polymers formed amyloid−polymer complexes with distinct morphologies. AS Fibrils Enhance UV Absorption and Fluorescence Emission of Conjugated Polymers after Interaction. To study the amyloid−polymer interactions, UV absorption and fluorescence spectroscopy was performed. Previously, UV absorption spectroscopy was performed to determine the binding of ligands to the protein.57 We assumed that if there is effective interaction of polymer with LMW and fibrillar AS, there might be a change in the absorption spectra of the polymers. UV absorption was measured in the range of 300− 500 nm for AS samples in the presence and absence of PF and PFS at the beginning of the incubation (soluble, LMW) and after AS fibrils formation. Only PFS control sample showed absorption maxima ≈ 340 nm, whereas PF alone sample showed a strong absorption at ∼370 nm (Figure 5A). However, 3780

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Figure 6. Effect of PF and PFS interaction with AS species. (A) CD spectroscopy showing PF and PFS did not alter the secondary structure of preformed fibrils (A), oligomers (B), and monomer (C). (D) ThT fluorescence showing unaltered ThT binding due to interaction of PF and PFS with different AS species. (E) SEC profiles of AS in the absence and presence of PF and PFS. (F and G) Tyr fluorescence of AS monomer (Ex 280 nm) and polymer fluorescence of the monomeric fractions (in the presence and absence of PF and PFS), respectively. (H) Polymer fluorescence in oligomer−PF and oligomer−PFS fractions isolated from size exclusion chromatography (SEC). AS oligomers only isolated from SEC were used as control.

state.58 The results of our UV and fluorescence studies (Figure 5) indicate that the electronic states of PF and PFS as free polymers are different from those after interaction with different AS species. The data suggest that both PF and PFS might interacted with the AS species, which inhibits the polymers self-aggregation. Complexation of PF and PFS with Preformed AS Oligomers and Fibrils without Alteration of AS Secondary Structure. To delineate the possible secondary structural alterations due to PF/PFS interaction with preformed AS fibrils, CD spectroscopy was performed with AS fibrils in the presence and absence of both polymers. The preformed AS fibrils were isolated by centrifugation of fibril solutions at 14 000g for 30 min and the pellet redissolved with buffer containing either PF and PFS such that the protein:polymers ratio becomes 1:1. The mixture was incubated for 1 h, and CD spectroscopy was performed. It was observed that in the presence of both polymers, the β-sheet signature of AS fibrils remained unchanged (Figure 6A). A similar study was also done with preformed AS oligomers (Figure 6B) and with monomer (Figure 6C) that was isolated from SEC. PF and PFS did not change the secondary structure of preformed fibrils, oligomers, as well as monomers (Figure 6A, 6B, and 6C). ThT fluorescence spectroscopy was also performed with these AS species both in the presence and in the absence of PF and PFS. No significant change in ThT fluorescence was observed

(Figure 6D) in the presence and absence of polymers, further suggesting that the major structural/amyloidogenic property of AS species remains intact in the presence of these charged polymers. Polymer Interaction with AS Monomers versus Preformed Oligomers. To study the extent of interaction of polymer to monomeric versus oligomeric species of AS in more detail, a SEC study was carried out for freshly dissolved AS in the presence and absence of PF and PFS. The SEC profile of AS alone showed two representative peaks: one at ∼7.5 mL (void volume) corresponding to oligomer elution and another at ∼15 mL corresponding to monomer elution (Figure 6E). The oligomers were isolated using a similar method that was used previously for many studies of AS and Aβ (associated with Alzheimer’s disease).15,23,44,45,47,48 In the presence of PF and PFS, the total eluted protein (as calculated from areas under the SEC peaks) was less than that of AS alone (Figure S4, left, Supporting Information). It is important to note that after dissolution of solid AS and subsequent mixing of PF and PFS the solution mixture was centrifuged to remove any aggregates that may interfere with the SEC column in this study. Therefore, there was a lower amount of eluted protein from the SEC study, indicating a higher amount of fibril formation immediately after addition of PF and PFS. Additionally, the interactions of monomeric and oligomeric species of AS with the polymers (PF/PFS) were performed by 3781

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fluorescence spectroscopy. A decrease in Tyr fluorescence at ∼305 nm by eluted AS monomers in the presence of both PF and PFS suggests that a lower amount of AS monomer was eluted compared to AS alone (Figure 6F and 6G), although the interaction of polymers to the AS monomer may also cause the observed decrease in Tyr fluorescence. This observation further supports both polymers accelerating the AS aggregation. When monomeric fractions were excited at 370 nm (excitation of PF), it showed characteristic fluorescence spectra of PF (Figure 6F), suggesting elution of PF occurs with the AS monomer. Similarly, AS monomers eluted with PFS also showed intense fluorescence with λmax at ∼420 nm (Figure 6G) after excitation at 340 nm, suggesting that PFS is also eluted with the AS monomer due to its binding to the AS monomer. To delineate the fluorescence property of bound polymers to the preformed oligomers, fluorescence studies were carried out with SEC isolated AS oligomer (Oligo) and AS-polymers (Oligo-PF or Oligo-PFS). AS oligomers (Oligo) alone did not show any significant fluorescence signal within the measured range of fluorescence (Figure 6H) as expected. In contrast, oligomers eluted in the presence of PF and PFS showed distinct fluorescence spectra, suggesting that the eluted oligomers were bound to the polymers (Figure 6H). Secondary Structure and Morphology of AS Oligomers Eluted in the Presence and Absence of Polymers. To further characterize the SEC isolated monomer and oligomer eluted in the presence and absence of each polymer, CD spectroscopy and EM studies were performed to elucidate the secondary structure and morphology, respectively. CD spectroscopy of eluted monomer and oligomers suggested that the presence of polymers does not affect the secondary structure of eluted AS monomer and oligomers (Figure 7A). Monomer in the presence and absence of both polymers mostly showed random coil conformation. However, the oligomers showed β-sheet conformation, irrespective of the presence and absence of polymers. To analyze the morphology of oligomers eluted in the presence and absence of both polymers, oligomers were isolated from SEC and the morphology was characterized using EM. The oligomers of only AS sample showed mixed populations of spherical oligomers, annular rings, and short wavy filaments under TEM (Figure 7B). The oligomers formed in the mixture of AS+PFS also displayed a very similar morphology, indicating that PFS did not alter the major morphological property of AS oligomers. Interestingly, the morphology of the oligomers isolated from the AS+PF mixture was significantly different from that of AS alone and AS+PFS mixture. Oligomers of AS+PF samples showed increased populations of small-sized oligomeric species with sparse population of annular ring-like structures (Figure 7B). These results suggest that the PF significantly alters the morphology of AS oligomers/protofibrils compared to PFS. Extent of Interactions of PF and PFS with Different Species of AS. To delineate the binding capacity of PF and PFS with different species of AS (monomers, preformed oligomers, and fibrils), polymer fluorescence was performed in the presence of different concentrations of various AS species (Figure 8A and 8B). The intensity of PF and PFS fluorescence (λmax at 418) was plotted against increasing concentration of different AS species (Figure 8C and 8D), and the dissociation constant (Kd) was determined. The data showed that the dissociation constant was lowest for PF−fibrils (2.55 ± 0.8 μM), suggesting that PF binds with fibrillar AS aggregates more strongly. Dissociation constant of PFS−fibrils were found to be

Figure 7. Secondary structure and morphology of AS oligomers isolated in the presence and absence of PF and PFS. (A) Circular dichroism spectroscopy of AS oligomers and monomers isolated from SEC in the presence and absence of both polymers did not show significant secondary structural changes. (B) EM images showing thin protofibrilar AS species along with some globular oligomers for AS sample alone. AS−PF oligomers showed small-sized oligomeric species. AS−PFS show protofibrils and globular oligomers similar to AS alone oligomers.

4.8 ± 0.75 μM. Similarly, both PF and PFS can interact with preformed oligomers moderately, and dissociation constants for PF−oligomers and PFS−oligomers were of 8.6 ± 1.1 and 11.3 ± 1.3 μM, respectively. Both charged polymers of PF and PFS interact with monomeric species less effectively. Dissociation constants of PF−monomer and PFS−monomers were found to be 13.6 ± 0.8 μM and 15.5 ± 1.05 μM, respectively. It was reported that amyloid formation is primarily driven by hydrophobic interactions between protein molecules.2,59 Although both PF and PFS contain charged groups in their side chains, both of them are also highly hydrophobic due to the presence of aromatic ring structures. The AS−polymer interaction could either be due to electrostatic interaction or be mediated through hydrophobic interaction. Since the monomeric/LMW state of AS is highly soluble with less overall hydrophobicity, the interaction between AS monomer and polymer could be mostly electrostatic in nature. The AS−PFS interaction could be due to electrostatic interaction between the positively charged N terminus of AS and the negatively charged side chains of PFS. In this context, the N terminus of AS has been shown to bind negatively charged phospholipids membrane.25 In contrast, negatively charged PFS is unlikely to bind the C terminus of AS because the C terminus contains mostly negatively charged amino acid residues. Interaction of PF with the N terminus (positively charged) is also not possible because of the same charge. Therefore, it is possible that PF may bind to the C terminus of AS, which is highly solvent exposed.60 The dissociation constant data further suggest that polymer binding is more to the ordered structure of AS species than that of disorder and highly charged monomer. The preferential bindings of polymers (PF and PFS) to AS oligomers and 3782

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Figure 8. Dissociation constant of different AS species and polymers. Fluorescence spectra of PF (A) and PFS (B) in the presence and absence of monomeric, oligomeric, and fibrillar AS. Different numbers are indicating the micromolar concentrations of different AS species. Changes in PF (C) and PFS (D) fluorescence at 420 nm due to addition of various concentration of AS monomer, oligomers, and fibrils.

Figure 9. Effect of salt in AS-bound PF/PFS fluorescence. NaCl (500 mM) significantly reduced the fluorescence intensity of PF/PFS that bound to the different AS species. Extent of reduction was maximum in the case of monomer and least for fibrils. Normalized PF and PFS fluorescence was plotted against different AS species.

preformed fibrils could be due to the hydrophobic interactions between AS species and polymers. To delineate the role of electrostatic interaction between binding of AS species and each of the polymers, monomeric, oligomeric, and fibrillar AS species were prepared as described before. PF and PFS binding to the AS species was performed in the presence and absence of 500 mM NaCl salt. The PF and PFS fluorescence in the presence of different AS species was decreased significantly when NaCl was present in the solution mixture. Salt-mediated reduction was more pronounced in the case of monomer compared to oligomers and fibrils (Figure 9). PF and PFS fluorescence when bound to the AS monomers were decreased by ∼60% and ∼55%, respectively, in the presence of NaCl. Oligomers bound PF and PFS fluorescence was decreased by

∼38% and ∼40%, respectively, in the presence of NaCl. However, for fibrils the reduction was only ∼21% and ∼27% for PF and PFS, respectively. The data signifies that for both polymers the electrostatic interaction between AS and polymers predominates in the case of monomers but hydrophobic and other interactions predominate for higher ordered aggregates like AS oligomers and AS fibrils. It is therefore possible that both polymers interact with AS monomer through electrostatic (mostly) and hydrophobic interaction (partially) and increase the local concentration of AS. This interaction eventually facilitates formation of aggregation competent oligomers, which subsequently convert to the fibrils. However, when each of the polymers binds to AS oligomers, it may increase and/or stabilize the aggregation 3783

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competent oligomers, resulting the faster aggregation kinetics of AS. Moreover, polymers binding to the AS fibrils and its subsequent stabilization may also lead to the faster fibrillation of AS. Furthermore, the different fluorescence intensity profiles of PF and PFS in the presence and absence of various AS species (monomers, oligomers, and fibrils (Figures 5 and 6) indicate that during aggregation the polymers constantly change their conformation to accommodate/bind different species of AS. PF and PFS Complexation Does Not Alter the Toxicity of AS Fibrils. To delineate whether PF and PFS modulate the cytotoxicity of AS fibrils, the cytotoxicity of fibrils formed in the presence and absence of polymers was evaluated using SHSY5Y cells. The MTT assay was performed for this purpose. The MTT assay is a widely used test to measure the potential cytotoxicity of compounds, wherein its inhibition of MTT reduction is determined.55 The presence of 5 μM AS fibrils resulted in 62% MTT reduction, suggesting that these AS amyloids were toxic to SH-SY5Y cells (Figure 10). PF and PFS

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ASSOCIATED CONTENT

S Supporting Information *

Table containing assignment of FTIR spectra, EM images, and amount of oligomers vs monomer in SEC. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge CSIR (37(1404)/10/EMR-11 to S.K.M.) and (09/080(0600)/2008-EMR-I to C.C.), DST (SR/ FR/LS-032/2009 to S.K.M.), DBT (BT/PR14344Med/30/ 501/2010 and BT/PR13359/BRB/10/752/2009 to S.K.M.), the Government of India for financial support, and the Central SPM Facility (IRCC, IIT Bombay) for AFM imaging and SAIF (IIT Bombay) for FTIR spectroscopy and electron microscopy. We also thank Sonali Maji for critical reading and suggestions and Mr. Prem Verma (Department of Physics, IIT Bombay) for the help during AFM imaging. D.G. acknowledges UGC (Government of India) for his fellowship.

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REFERENCES

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Figure 10. Toxicity of AS fibril−polymer complex. Fibrils formed in the presence and absence of PF and PFS showing almost similar toxicity. AS monomer was used as control and showed no toxicity.

were moderately cytotoxic as they showed an MTT reduction of ∼85%. However, AS fibrils formed in the presence of each polymer (amyloid−polymer complex) showed almost MTT reduction (65%) similar to AS fibrils alone samples. Data indicate that PF and PFS did not alter the toxicity of AS fibrils. Monomeric AS (isolated from SEC) was used as a control, and it did not show any cytotoxicity. Data suggest that although both PF and PFS modulate AS aggregation and produce fibrils with different morphologies, the cytotoxicity of resultant fibrils remained unaltered.

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CONCLUSION Our results suggest that the water-soluble polymers PF and PFS carrying a positive or negative charge, respectively, in their side chains accelerated the AS aggregation significantly. Both polymers were bound to fibrils more efficiently than the oligomers and bind to the lowest extent to monomers, suggesting that binding to amyloidogenic oligomers and/or fibrils by polymers could cause the faster rate of fibrillation. We suggest that the hydrophobicity of the polymer backbone is the major driving force for the AS oligo/fibrils−polymer interaction and therefore polymer-mediated AS aggregation. Both polymers however did not alter the major secondary structure of the oligomers and fibrils, even after incorporation into the fibrils. The increased fluorescence of the conjugated polymers is possibly due to incorporation of polymers into higher order fibrils. 3784

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dx.doi.org/10.1021/la404739f | Langmuir 2014, 30, 3775−3786