Disassembly and Reassembly of Amyloid Fibrils in Water−Ethanol

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Disassembly and Reassembly of Amyloid Fibrils in Water-Ethanol Mixtures Sophia Jordens, Jozef Adamcik, Idit Amar-Yuli, and Raffaele Mezzenga* ETH Zurich, Food & Soft Materials Science, Institute of Food, Nutrition & Health Schmelzbergstrasse 9, LFO E23, 8092 Zu¨rich, Switzerland Received September 20, 2010; Revised Manuscript Received November 12, 2010

This work presents the structural analysis of amyloid-like β-lactoglobulin fibrils incubated in ethanol-water mixtures after their formation in water. We observe for the first time the disassembly of semiflexible heat-denatured β-lactoglobulin fibrils and reassembly into highly flexible wormlike fibrils in ethanol-water solutions. Tapping mode atomic force microscopy is performed to follow structural changes. Our results show that in addition to their growth in length, there is a continuous nucleation process of new wormlike objects with time at the expense of the original β-lactoglobulin fibrils. The persistence length of wormlike fibrils (29.43 nm in the presence of 50% ethanol), indicative of their degree of flexibility, differs by 2 orders of magnitude from that of untreated β-lactoglobulin fibrils (2368.75 nm in pure water). Interestingly, wormlike fibrils do not exhibit a multiple strands nature like the pristine fibrils, as revealed by the lower maximum height and the lack of clear height periodicity along their contour length profile. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrates that the set of polypeptides obtained by ethanol degradation differs in some fractions from that present in pristine β-lactoglobulin fibrils. ATR-FTIR (attenuated total reflectance-Fourier transform infrared) spectroscopy also supports a different composition of the secondary structure of wormlike fibrils with a decreased amount of R-helix and increased random coils and turns content. These findings can contribute to deciphering the molecular mechanisms of protein aggregation into amyloid fibrils and their disassembly as well as enabling tailormade production of protein fibrils.

Introduction One of the main foci of the research in the field of protein aggregation is to comprehend the formation of amyloid fibrils and to elucidate fully their structural characteristics. These structures are filamentous aggregates of proteins that have been known to cause, or be involved in, diseases such as Parkinson’s, Alzheimer’s, Creutzfeldt-Jakob disease, type II diabetes, and bovine spongiform encephalopathy (BSE) because of the formation of insoluble extracellular deposits.1-4 Even though the proteins involved in these diseases seem to have little in common among each other, there exist similarities in the fibrillar structures they form. Furthermore, the ability to aggregate into fibrils is, at least in vitro, not limited to proteins associated with such diseases:1 the formation of highly organized amyloid-like aggregates has been found to be a generic property of polypeptides.5-7 Protein aggregation is one of the major challenges in biotechnological and pharmaceutical processes.8 In addition, some of the fibrils may even have promising applications as macromolecular assembly-based bionanomaterials9 and as weight-effective thickeners or texturizers for foods.10 The proteins that could be considered for food applications are whey, egg white, and soy proteins, all of which have been shown to be capable of fibrillation.11 By taking advantage of soft condensed matter approaches and applying them to complex food systems, it is conceivable to also use these structures as convenient building blocks for functional foods.12 It has been argued that prefibrillar aggregates, such as protofibrils that only exist transiently, are an important factor * To whom correspondence [email protected].

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in the pathogenesis of amyloids.13,14 This includes rigid protofilaments, which aggregate in a later stage into multistranded fibrils15 and protofibrils with a beaded structure.14 Even though their assembly and detailed molecular mechanisms of fibrillation have not yet been fully elucidated,7,13,16 it is clear that stable and insoluble fibrils result from proteinaceous selfassembly: the misfolding or partial unfolding of globular proteins under favorable physicochemical conditions such as high temperature, low pH, or both is often followed by aggregation into mature long, stiff, fibrillar aggregates.9,17 This is, however, strongly determined by a delicate balance of hydrophobic and electrostatic interactions.11,18 According to recent findings, fibrils are not formed of the whole monomer but rather only a defined set of peptides present in the original protein.16,19 Protein fibrils are rich in β-sheets with the polypeptide backbone arranged perpendicular to the fibril axis, resulting in a cross-β arrangement with high internal order.20-22 Fibrils are typically composed of two or more helically arranged protofilaments with a diameter of approximately 2-10 nm and are usually >1 µm in length.18,23 The periodicity of the structure depends on the number of protofilaments present in a single fibril.18 Understanding amyloid fibril formation and degradation will be highly valuable and may assist in dealing with the aforementioned diseases as well as developing new technological applications. β-Lactoglobulin, the protein used in this work, is the major whey protein in bovine milk and therefore of high importance to the food industry.24 Given the small molecular weight of 18 400 Da25 and globular nature, it has proven to be a well-suited model system for studying fibrillar protein aggregation.18,26,27

10.1021/bm101119t  2011 American Chemical Society Published on Web 12/10/2010

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Ethanol has been shown to induce fibril formation in proteins, including native β-lactoglobulin, by changing the hydrational forces and promoting protein unfolding, and as such, it can be viewed as a valid alternative to heat denaturation.26,28-31 This solvent decreases the polarity of the system and this accompanied by the weakening of the stabilizing hydrophobic interactions, hence shifting the equilibrium toward the denatured state.32 As a result, this conformational modification facilitates protein denaturation, aggregation, and therefore fibril formation.29,33,34 The fibrils formed in the presence of ethanol are described as being wormlike, short, and flexible with a “stringof-beads” structure and thinner than those formed by heat denaturation at a low pH.26 In the present work, to understand the effects of a polar protic solvent on amyloidal fibrils already formed, we incubated dialyzed β-lactoglobulin fibrils with different concentrations of ethanol. This procedure differs substantially from previous works, which use ethanol already in the stage of denaturation of the native protein prior to fibrillation: here ethanol has the primary role of a disrupting agent of semiflexible protein fibrils already formed in water. High-resolution atomic force microscopy was used as the main tool for structural analysis: contour and persistence length proved to be powerful means of tracking structural changes in the samples over time and measure their size distribution. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and attenuated total reflectanceFourier transform infrared spectroscopy (ATR-FTIR) were used as further methods to gain insight into the internal structure of amyloid-like fibrils under the influence of ethanol. We show, for the first time, that ethanol not only promotes protein unfolding and denaturation but also can be used as a means to disassemble amyloid fibrils previously formed in water and promote reassembly of different peptide segments into a new type of amyloid fibrils with different structural features.

Experimental Details Amyloid Fibril Formation. The primary dialysis and fibril formation were carried out according to available literature reports.18,27 We prepared 100 mL of a 10% w/w β-lactoglobulin solution by dissolving 10 g of BioPURE-β-lactoglobulin (Davisco Foods International) in deionized water (Milli-Q purification system, Millipore) and setting the pH to 4.6. Centrifugation for 15 min at 15 000 rpm allowed us to remove aggregates or denatured protein particles. The supernatant was collected, and the pH of the solution was adjusted to 2, followed by vacuum filtration using a pore size of 0.22 µm (Millipore Corporation). Dialysis tubing (6000-8000 MWCO, Spectrum Laboratories) was placed in 500 mL of a 1 mM EDTA solution at 4 °C. Prior to filling them with the β-lactoglobulin solution, the tubes were rinsed with distilled water. The clamped tubes were placed in 4 L of Milli-Q at pH 2 and stored at 4 °C while keeping the water in constant motion with a magnetic stirrer. The water bath was then changed periodically with pH 7 Milli-Q water. A significant increase in the pH of the used water bath indicated a slowing down of ion exchange and therefore a low residual ion concentration inside the tubes. After the protein solution was collected, its pH was readjusted to 2. A second filtration was carried out using a 0.45 µm Millipore filtering syringe, after which the protein concentration was determined by dry mass upon complete evaporation of the liquid (measurement averaged over three aliquot measurements). The solution was then diluted to 2% w/w. A Schott flask containing the dialyzed protein solution was placed in an oil bath at 90 °C. Both the oil and the solution were kept in motion with magnetic stirrers for 5 h. The fibril solution was then removed from the bath and cooled immediately. The conversion rate of β-lactoglobulin monomers into fibrils was 75%.35 The fibril solution was dialyzed again against pH 2 Milli-Q

Jordens et al. water for 7 days using 100 000 MWCO dialysis tubing (Spectrum Laboratories) to remove unreacted protein and low-molecular-weight residual peptides. Atomic Force Microscopy (AFM) Imaging.18,36 A volume of 20 µL of the sample to be imaged was pipetted onto a freshly cleaved mica disk with a diameter of 9.9 mm (Ted Pella) and left to adsorb for 2 min at room temperature. After rinsing with Milli-Q water and drying with clean air the sample was topologically analyzed with a Nanoscope V scanning probe microscope (Tip model TAP150A; Veeco Instruments) in tapping mode at a scan rate of 1 Hz. Incubation with Ethanol. All experiments were performed with a final β-lactoglobulin fibril concentration of 0.1% w/w, and the pH was readjusted to 2, if needed, in all samples. Solutions containing 0, 10, 20, 30, 40, or 50% ethanol absolute (analytical grade, ACS, Scharlau S.L.) were incubated at 37 °C. Contour Length. The contour length of the fibrils was determined by analyzing AFM images with the program Ellipse36 allowing the tracing of fibril coordinates. The contour length can then be calculated from these xyz coordinates using Mathematica (Wolfram Research): The algorithm is based on splitting each fibril into small, rigid segments, whose total collective length equals to the contour length of the fibril. The total number of counts was 1703 for wormlike and 1183 for semiflexible fibrils. Persistence Length. The persistence length can be derived from the decay of the bond correlation function for semiflexible polymers, 〈cos θ(s)〉 ≈ exp(- s/2lp), where θ is the angle between the tangents at two positions on the polymer chain placed at a contour length distance s.18 The result is then averaged over the total contour length of the object. 115 wormlike and 301 rigid fibrils were analyzed. SDS-PAGE. The samples were mixed with 2× SDS sample buffer and dithiothreitol (DTT) according to the literature37 before they were denatured at 100 °C for 10 min. The solutions along with an unstained protein molecular weight marker (Fermentas) were separated in a Criterion 18% Tris-HCl precast gel (Bio-Rad Laboratories) at a voltage of 100-200 V for a total running time of 1.5 h. The gel was stained with 0.1% Coomassie Brilliant Blue R-250. We emphasize that the fibrils were not exposed to any reducing environment prior to the measurement. ATR-FTIR. Freshly prepared and 3 month old 50% ethanol-water solutions containing 0.1% β-lactoglobulin fibrils were concentrated 10fold by centrifugal ultrafiltration (Vivaspin 500, sartorius Stedim Biotech S.A.). The measurements were conducted with a Bruker Alpha-P spectrometer (Bruker Optik, GmBH), equipped with a single reflection diamond ATR sampling module, at a resolution of 2 cm-1, and the spectra were averaged over 50 scans using the Bruker Opus software. Multi-Gaussian fitting was utilized to resolve individual bands in the spectra. The peaks were analyzed in terms of peak frequencies, width at half-height, and area. The samples’ spectra were backgroundsubtracted against the appropriate water control spectra to resolve the measured amide I′ band of β-lactoglobulin at 1700-1600 cm-1. The amide I′ band was then resolved by second-derivative Savitzky-Golay.

Results and Discussion Figure 1 represents the AFM images of β-lactoglobulin fibrils incubated at 37 °C in 0, 40, and 50% ethanol-water solutions for different time periods. The presence of short wormlike structures was observed in the samples containing >30% ethanol already after 1 week of incubation. In 10 and 20% ethanol-water mixtures (data not shown), no difference was observed compared with the original semiflexible fibrils. The images also indicate that with longer incubation time the contour length of wormlike structures increases, whereas the amount of original fibrils decreases in mixtures with high ethanol concentrations. Several distinct and striking differences between these novel wormlike structures and the typical original fibrils can be clearly observed (Figure 2, left): they not only are more flexible but

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Figure 1. AFM images of β-lactoglobulin fibrils at 0.1% w/w concentration treated with (a) 0, (b) 40, and (c) 50% ethanol after 0, 1, 5, and 8 weeks at 37 °C. The incubation time (weeks) is indicated in the top right corner of the first row (valid for all three rows). The scale bars in the bottom right image apply to all images.

Figure 2. AFM image of β-lactoglobulin fibrils at 0.1% w/w treated with 40% ethanol after 5 weeks of incubation at 37 °C showing flexible structures in the presence of normal amyloid fibrils (left) and a 3D representation of the same scan (right). We can obtain the height of the objects by subtracting the height of the background (12 nm; indicated by the arrow) from the heights read on the scale bar.

also exhibit a lower average height of 2 to 2.5 nm (Figure 2, right) compared with that of original fibrils, which has been found to be around 4, 6, 8, or 10 nm depending on the number of filaments in one fibril.18 Additionally, there is no evidence of a “string-of-beads” appearance. Single-molecule imaging with AFM further reveals that the small wormlike fibrils are not composed of multistranded filaments. Figure 3 indeed shows that the periodic fluctuations in the height profile typically found along pristine β-lactoglobulin fibrils18 are not present in these wormlike structures, which is the first criterion for screening the presence of multistranded fibrils. This is also in line with the lower maximum height observed for the wormlike fibrils compared with pristine β-lactoglobulin fibrils shown in Figure 2. The clear difference in flexibility of the two types of structures is reflected in their pronounced difference in persistence length, lp. The calculation is performed as described in the Experimental Details section. Low lp corresponds to high flexibility of the polymer. A typical calculated average value of the persistence length for untreated β-lactoglobulin fibrils is 2368.75 nm after 1 week of incubation at 37 °C, whereas the wormlike objects found in 50% ethanol-water solutions have lp ) 29.43 nm,

which is about two orders of magnitude less. Figure 4 shows the fit of the bond correlation function with the exponential decay yielding the persistence length. To provide further insight into the mechanisms leading to the formation of the flexible wormlike fibrils, we have counted the average number of objects and their total contour length per unit surface area: the results of this statistical analysis are shown in Figure 5. The trends in this Figure confirm the observation that the number and total contour length of the wormlike structures increase over time, in the case of both the 40 and 50% ethanol-water solutions. Because here the total contour length is normalized for 1 µm2, it represents a direct, equivalent measure of the total mass (per unit area) of the flexible structures, confirming that this quantity increases steadily during the whole observation time of 8 weeks. Similarly, it can be concluded from the increase in the density number of wormlike objects that the nucleation process occurs continuously during the same time. The wormlike structures must grow at the expense of the pristine long amyloid fibrils because the initial dialysis of the freshly formed semiflexible amyloidal fibrils in water removed all of the unreacted protein or the hydrolyzed fragments thereof.

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Figure 3. Height profile analysis of (a) an untreated β-lactoglobulin fibril (0% ethanol) and (b) a wormlike object obtained by incubation with 40% ethanol at 37 °C for 5 weeks. The height along the fibril is plotted versus the contour length of the analyzed section.

Figure 4. Decay of 〈cosθ(s)〉 as a function of the contour length s for rigid β-lactoglobulin fibrils incubated at 37 °C in the absence of ethanol for 1 week (O) and for the wormlike β-lactoglobulin structures incubated at 37 °C with 50% ethanol (b). The exponential fit results in lp,rods ) 2368.75 nm (dashed) and lp,flexible ) 29.43 nm (solid).

This is indeed evident from Figure 5, where the area-normalized total contour length of the long semiflexible original fibrils (and therefore their total mass per unit area) decreases with incubation time from 0 to 8 weeks. This change is larger in the mixtures containing 50% ethanol, with the total contour length per unit area (635 nm) being almost half of that in the 40% ethanol solution (1079 nm) after 8 weeks of incubation. Because both the nucleation and growth processes of these wormlike fibrils occur steadily and continuously, the average contour length (total contour length/numbers of fibrils) is not the most informative structural parameter, and a contour length distribution provides more valuable insight. This is shown in Figure 6 and immediately indicates a clear shift toward longer contour lengths of the wormlike amyloid fibrils during incubation time. The contour length of the vast majority of the measured structures does not exceed 550 nm, even after 8 weeks

of incubation, and is therefore much shorter than the original fibrils (1-10 µm).23 The maximum in the distributions (most probable contour length) shifts from 75 to 125 nm and from 75 to 175 nm when going from 1 to 8 weeks incubation for the 40 and 50% ethanol solutions, respectively. Our results from SDS-PAGE are in accordance with the literature16 in that the fibrillar aggregates of β-lactoglobulin formed by heat denaturation could be broken down into constitutive building blocks (Figure 7). Indeed, all samples containing fibrils produced bands at lower molecular weight levels than the β-lactoglobulin monomer (18.4 kDa), supporting the hypothesis that the fibrils are formed of peptides and not the whole monomer. During incubation with ethanol, the disassembly of β-lactoglobulin fibrils yields at least one new peptide of a molecular weight around 12 kDa (see band in lanes 2, 3, 5, and 6 in correspondence of the arrow in the markers lane M) regardless of the presence or absence of unreacted proteinaceous material in the solution. This suggests that the building blocks of the flexible structures that are formed during incubation with ethanol differ slightly from the set of peptides in the original fibrils. ATR-FTIR was used to examine the impact of prolonged incubation in ethanol on the secondary structure of the β-lactoglobulin fibrils. This spectroscopy technique is sensitive to, and commonly used for, monitoring R-helix-to-β-sheet refolding that accompanies protein aggregation. The peaks’ positions in the IR amide I′ band region (1700-1600 cm-1) and relative content of the secondary structure elements for the fresh fibrils in 50% ethanol-water mixtures and after 3 months of incubation at 37 °C are listed in Table 1. Prolonged exposure to ethanol resulted in some loss of the secondary structure, which is expressed as a partial transformation from R-helical into turns and random coils unordered structures. The relative R-helix content decreased by 32%, whereas the relative amount of turns and random coils both increased by 16%. Furthermore, the β-sheets remained intact despite the long incubation. Control experiments at the same incubation time in pure water were

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Figure 5. Change in the density number of wormlike fibrils (number per µm2; b) as well as the total mass, expressed as total contour length per µm2, for the wormlike structures (O) and original fibrils ([) over time for 0.1% w/w β-lactoglobulin fibrils incubated in (a,b) 40 and (c,d) 50% ethanol.

Figure 6. Evolution of contour length of flexible structures found in solutions of 0.1% w/w β-lactoglobulin fibrils incubated with (a) 40 and (b) 50% ethanol. The contour length distribution is shown for 1 (light blue), 5 (medium blue), and 8 weeks (dark blue) of incubation at 37 °C.

also carried out to reinforce the findings that these changes are solely due to the presence of ethanol. The secondary structure

Figure 7. Analysis by SDS-PAGE of 0.1% w/w dialyzed β-lactoglobulin fibril solutions (lanes 1-3), undialyzed β-lactoglobulin fibril solutions (lanes 4-6), and native β-lactoglobulin (lane 7) incubated at 37 °C for different time periods and varying ethanol concentrations. Molecular weight markers (M, kDa) 116.0: β-galactosidase, 66.2: BSA, 45.0: ovalbumin, 35.0: lactate dehydrogenase, 25.0: REase Bsp98I, 18.4: β-lactoglobulin, 14.4: lysozyme. (1) 0% ethanol, 1 h; (2) 40% ethanol, 4 months; (3) 50% ethanol, 4 months; (4) 0% ethanol, 5 months; (5) 40% ethanol, 5 months; (6) 50% ethanol, 5 months.

of the highly flexible wormlike fibrils present in ethanol-water mixtures portrays higher contents of unordered structure, that is, random coils and turns, compared with the pristine elongated

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Figure 8. Schematic representation of the formation of amyloid fibrils from a globular protein during heat denaturation, hydrolysis under acidic conditions, and the subsequent disassembly/reassembly during exposure to ethanol. Table 1. Secondary Structure Content of 50% Ethanol-Water Solutions Containing 1% w/w β-Lactoglobulin Fibrils after 0 and 3 Months of Incubation at 37 °C As Determined by ATR-FTIR area % conformation

wavelength [cm-1]

0 days

3 months

β-sheet random coil R-helix turn

1613-1637 1637-1646 1646-1662 1662-1682

30 36 22 12

29 42 15 14

fibrils, which is highly consistent with the observation of the greatly reduced persistence length. Contrary to previous opinions38-41 it is now believed that the partially unfolded protein initially undergoes acid hydrolysis, yielding peptides, which then aggregate to form amyloid fibrils held together by intermolecular β-sheets. The constituting peptides of these fibrils can differ from the building blocks of the original protein.16,19 Our results are in line with these findings. Upon exposure to an organic solvent like ethanol, a disassembly of fibrils into smaller, wormlike structures is observed. This may be caused by a change in solubility and denaturation behavior of the system due to the decreased dielectric constant. The temporal evolution of this rearrangement can be attributed to the above-mentioned modified solvent conditions and consequently the changed total free energy in the system: the system is forced to evolve structurally toward equilibrium to minimize the total free energy. A schematic drawing of the process is shown in Figure 8. Finally, it is noteworthy to highlight that the occurrence of a highly flexible structure for the wormlike fibrils together with their single-stranded nature are likely to be two factors directly connected with each other and not independent. By comparison, single-stranded β-lactoglobulin fibrils (or protofilaments) obtained by heat denaturation of β-lactoglobulin in water are known to have not only a maximum height of 2 nm (and thus, identical to that observed for the wormlike fibrils studied in the present work) but also a much greater persistence length of ∼1 µm and a strong tendency to form multistranded mature β-lactoglobulin fibrils.18 It is then straightforward to rule out fibrils “width” as the main cause of flexibility/rigidity. Furthermore, in very recent work, it has been shown that it is the liquid crystalline nature of the rigid β-lactoglobulin protofilaments that is the driving force responsible for the alignment of the individual strands in multistranded β-lactoglobulin fibrils.15 In the present case, multistranded fibrils cannot develop because wormlike fibrils are way too flexible (30 times more than β-lactoglobulin protofilaments based on persistence length consideration!) to allow liquid crystalline interactions. This particular case, although very specific, illustrates well the importance of structural analysis of amyloid fibrils as a first step toward the unraveling of their formation mechanisms.

Conclusions We have shown that multistranded β-lactoglobulin fibrils generated in water at high temperature (90 °C) and low pH (2) can be disassembled from their original form and reassembled into a different type of amyloid fibril by simply exposing and incubating them in ethanol-water mixtures. These wormlike fibrils are single-stranded, shorter (contour length of