Article pubs.acs.org/Biomac
Self-Assembly of Ovalbumin into Amyloid and Non-Amyloid Fibrils Cecile Lara, Simon Gourdin-Bertin, Jozef Adamcik, Sreenath Bolisetty, and Raffaele Mezzenga* Food and Soft Material Science, Institute of Food, Nutrition and Health, ETH Zürich, Schmelzbergstrasse 9, LFO E 23, 8092 Zürich, Switzerland S Supporting Information *
ABSTRACT: We study the fibrillation pathway of ovalbumin protein and report the simultaneous formation of several types of fibrils, with clear structural and physical differences. We compare the fibrillation mechanisms at low pH with and without salt, and follow the kinetics of fibrils growth by atomic force microscopy (AFM), static and dynamic light scattering (SLS, DLS), and small-angle X-ray scattering (SAXS). We show that, among the morphologies identified, long semiflexible amyloid fibrils (type I), with persistence length Lp ∼ 3 μm, Young's modulus E ∼ 2.8 GPa, and cross-β structure are formed. We also observe much more flexible fibrils (type III, Lp ∼ 63 nm), that can assemble into multistranded ribbons with time. They show significantly lower intrinsic stiffness (1.1 GPa) and a secondary structure, which is not characteristic of the well-ordered amyloids, as determined by circular dichroism (CD), wide-angle X-ray scattering (WAXS), and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). In between these two main classes of fibrils, a third family, with intermediate flexibility (type II, Lp ∼ 300 nm), is also resolved.
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amyloid fibrils assembled from other globular food proteins such as dialyzed β-lactoglobulin or lysozyme.19−21 This class of micrometers-long semiflexible and rigid fibrils, with high βsheet content, is identified for the first time from the ovalbumin protein, and referred in what follows as type I fibrils. Among the various types of fibrillar aggregates, a third main morphology (type II), with intermediate thickness and flexibility, is also resolved. The fibrillation process depends on several parameters, such as the pH, which affects the protein net charge and hydrolysis upon heat treatment,22 and the ionic strength of the solution, with counterions influencing the proteins electrostatic interactions.13 We therefore compare the kinetics of fibrils formation in two conditions: with and without addition of 50 mM NaCl. The distribution of the fibrils contour lengths and their persistence lengths, at the two conditions of low and high ionic strength, as well as their Young's modulus distribution were determined by AFM images analysis and AFM peak force nanoindentation, respectively. Scattering techniques such as small-angle X-ray scattering (SAXS), wide-angle X-ray scattering (WAXS), and dynamic and static light scattering (DLS, SLS) were used to characterize further the structural features in solution or in the dry state. Mass spectrometry (MALDI MS) and gel electrophoresis (SDS-PAGE) were also used to get a better understanding of the ovalbumin self-assembly process at the peptide sequences length scale. Finally, Fourier transform infrared spectroscopy (FTIR) and circular dichroism (CD) gave evidence of the secondary structure differences between the two extreme types of fibrils: type I (semiflexible) amyloids and type III (worm-like) flexible fibrils.
INTRODUCTION Protein aggregation is one of the most challenging fields in biology and medicine because a large number of human and animal diseases are related to protein self-assembly into amyloid fibrils.1−3 Non-disease-related amyloids, however, are also of interest in other fields. In food science and technology, for example, protein fibrils offer desirable properties, especially for their interfacial and texture building features.4−6 In materials science, they provide functional and mechanical properties with applications ranging from medicine to electronics.7−10 Ovalbumin is one of the most important protein components in egg white and has multifunctional properties, such as its ability to foam and to form gels upon heating.11,12 The native protein consists of 385 amino acid residues, has a molecular weight of 44500 Da, an isoelectric point of 4.5 and a denaturation temperature of 84 °C at pH 7.6 Under specific denaturation conditions, such as heat treatment around 75−80 °C, at pH 2 or 7, ovalbumin has been shown to form fibrils in vitro. The fibrils are always reported to have a very flexible morphology with a contour length ranging between 25 and 300 nm,13−16 depending on the experimental conditions such as pH, temperature, ionic strength, and so on. The fibrillar networks have been shown to undergo gelation, above the critical concentration, for given pH and ionic strength.17 In some studies, a decrease of α-helical content, binding of thioflavin T to the ovalbumin aggregates,17 or of Congo red,18 suggest the formation of β-sheet type of structures. In this work, however, we combine single molecule microscopy technique with bulk scattering and spectroscopic techniques, to demonstrate that, at pH 2 and 90 °C, ovalbumin does not only form the very flexible fibrils morphology reported so far (here referred as type III), but also a longer and stiffer type of fibrils is formed. Only this latter class of fibrils exhibits the typical fingerprint of amyloid fibrils, in direct analogy with © 2012 American Chemical Society
Received: September 20, 2012 Revised: October 24, 2012 Published: October 25, 2012 4213
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Figure 1. AFM height image (a) and height profile along the black arrow (b) of ovalbumin fibrils after 24 h heat treatment (no salt added). The scale bar represents 1 μm. In the inset, the persistence length values are given for the three types of fibrils, designated by I, II, and III on the image. The three corresponding heights statistical average are attributed to the respective peaks in the profile.
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1.542 Å) source, collimated by three pinhole (0.4, 0.3, and 0.8 mm) collimators, the applied voltage and filament current being 45 kV and 0.88 mA, respectively. The data were collected either by a twodimensional argon-filled detector (for SAXS) or with the help of a Fuji Film BAS-MS 2025 imaging plate system: 15.2 × 15.2 cm, 50 μm resolution (for WAXS). The samples for SAXS, 0.1 w/w % protein solutions, were transferred to 1.5 mm quartz X-ray capillaries, while protein powders were used and held between mica plates for WAXS experiments. The azimuthally averaged scattered intensities versus the scattering vector q, with q = (4π/λ)sin θ (where 2θ is the scattering angle) for SAXS analysis, were fitted by the flexible cylinder with polydisperse contour length model (see Supporting Information).23,24 Gel Electrophoresis (SDS PAGE). For each sample, 8 μL fibril aliquots at 2 mg/mL were mixed with 2 μL of dithiothreitol (DTT) and 10 μL of loading buffer (100 mM Tris-HCl, 4 w/v % SDS, 0.2 w/v % bromophenol blue, 20 w/v % glycerol). The solutions were then heated 5 min at 95 °C on a T3000 Thermocycler. The 20 μL solutions and the molecular weight (Mw) markers (broad range, BioLabs No. P7702S) were loaded on Criterion precast gels 16.5% Tris-Tricine/ Peptide 1.0 mm (Bio Rad Laboratories) and separated at 100 V. The gels were then stained (0.1 w/v % Coomassie Brilliant Blue R-250, 10% acetic acid, 40% dd H2O, and 50% methanol) and destained overnight. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS). The fibrils were separated by ultracentrifugal filtration with Micosep Advance Centrifugal Device with 0.45 μm Supor Membrane. The efficacy of the separation of the different types of fibrils was verified by AFM imaging. To remove the remaining nonfibrillar material and protein fragments from the fibrils solutions, a Micosep Advance Centrifugal Device with a 100 K MWCO membrane was used. Several centrifugation steps were run at 3000 rpm and 20 °C until no protein was detected in the filtrate by UV−vis spectroscopy. The samples were 5-fold diluted in 1 v/v % TFA and then desalted using a C18 ZipTip (Millipore, MA, U.S.A.), eluted with 2 μL matrix solution (α-cyano-4-hydroxycinnamic acid (10 mg/mL) dissolved in 50 v/v % acetonitrile/0.5 v/v % TFA), dried, and analyzed by MALDIMS (MS/MS) in the positive-ion mode. The measurements were performed on the MALDI mass spectrometers Ultraflex II and Autoflex II (both from Bruker, Germany). Based on the masses and the corresponding MS/MS data, the most probable amino acid (AA) sequences and the cleavage sites for some peptides were proposed. Far-UV Circular Dichroism (CD). Circular dichroism data were recorded using a Jasco J-815 spectrometer, equipped with a Peltiercontrolled cell holder. Spectra at 20 °C were collected using a precision quartz cell of 2 mm path length from 190 to 250 nm with a bandwidth of 1 nm and a scan speed of 50 nm/min. All spectra were recorded after diluting the incubated 2 wt % ovalbumin aliquots 100× in Milli-Q water. Spectra were background subtracted, averaged over three scans, and smoothed by OriginPro 8G. The percentages of αhelical and β-sheet contents were determined with CDPRO (continll, selcon3, and cdsstr analysis), based on a reference set of 56 proteins in the 190−240 nm wavelength range.
MATERIALS AND METHODS
Fibrils Preparation. Ovalbumin from chicken egg white, obtained from Sigma (A 5503), was dissolved in pH 2 Milli-Q water, dialyzed for one week at 4 °C against Milli-Q water (Spectra Por molecular porous membrane tubing MWCO 6−8 kDa), and then freeze-dried. The purified ovalbumin powder was dissolved at 2 w/w % into pH 2 Milli-Q water, with and without 50 mM NaCl (Sigma, ≥99.5%), and the pH was then adjusted to 2 with 1 M HCl. Protein solutions were introduced in an oil bath preheated at 90 °C and stirred during the incubation process. Aliquots were taken at given heating times ranging from 1 to 265 h, quenched in ice-cold water and stored at 4 °C. Atomic Force Microscopy (AFM). Aliquots were diluted to protein concentrations ranging from 10−5 to 10−1 w/w % with pH 2 Milli-Q water, and 20 μL were incubated for 2 min on freshly cleaved mica, rinsed with Milli-Q water, and dried with air. Images were collected using a Nanoscope VIII Multimode Scanning Force Microscope (Bruker) operated in tapping mode in air. Images were flattened using the Nanoscope 8.10 software, and no further image processing was done. The coordinates of fibrils on the substrate were acquired using Ellipse software and further processed with Mathematica (Wolfram Research) to extract information such as height, period, contour length; the persistence length was extracted from the exponential decay of the bond correlation function, as discussed previously.20 Statistics were done on a significant number of fibrils (from 150 to 1500 fibrils were analyzed for each morphology at the different times). Peak Force Quantitative Nano Mechanical AFM (PF QNM). PF QNM measurements were performed with a MultiMode VIII Scanning Probe Microscope (Bruker), operated in intermittent mode under ambient conditions, at a scan rate of 1 Hz. The microscope was covered with an acoustic hood to minimize vibrational noise. The AFM probe was calibrated on the calibration samples such as lowdensity polyethylene (LDPE) and polystyrene (PS), covering ranges of moduli going from 100 MPa to 2 GPa for LDPE and from 1 to 20 GPa for PS. The analysis of the Derjaguin−Mueller−Toporov (DMT) modulus was performed with the software Nanoscope Analysis. Dynamic and Static Light Scattering (DLS, SLS). A LS Instruments device, equipped with He−Ne Laser, emitting a polarized light beam of wavelength of 632.8 nm was used for the static and dynamic experiments. The measurements were done on filtered (0.22 μm cellulose acetate syringe filters) and unfiltered fibrils. The dynamic light scattering measurements were performed at a fixed angle of 90° by averaging 3 runs of 600 s each. In dynamic light scattering, the time correlation function (TCF) of the scattered intensity was analyzed by the CONTIN method. The static light scattering measurements were performed with an angle ranging from 40 to 140° by steps of 10°. The radius of gyration (Rg) was calculated from the Guinier approximation of the form factor (see Supporting Information, Figure S1). Small Angle X-ray Scattering (SAXS) and Wide Angle X-ray Scattering (WAXS). X-ray scattering experiments were performed on a MicroMax-002+ microfocused beam with a sealed tube Cu Kα (λ = 4214
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Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR). FTIR spectra were obtained with a Varian 640 FTIR Spectrometer equipped with a Specac Diamond ATR Golden Gate. Samples powders were scanned over the range of 4000 to 600 cm−1 with a resolution of 4 cm−1 at room temperature and averaged over 64 scans. The measured amide I band of ovalbumin in the 1700−1600 cm−1 range was then resolved by second-derivative analysis and peak deconvolution.
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RESULTS AND DISCUSSION After 24 h heat treatment of the dialyzed ovalbumin at 90 °C in pH 2 Milli-Q water, three kinds of fibrils were observed. As can be seen in the AFM height image given in Figure 1, their structural differences allow for immediate distinction between the fibrils. According to the height profile, taken along the black arrow in Figure 1, the three types of fibrillar structures can first be differentiated by their height values. A statistical analysis shows that the thicker, medium and thinner fibrils have an average diameter (height) of 7 ± 2, 4 ± 1, and 2.5 ± 1 nm, respectively. Second, their flexibilities differ by 1 order of magnitude between thick (type I) to medium (type II) and medium to thin (type III): their respective persistence lengths, obtained by the decay of the bond correlation functions,20,25 are given in Figure 1, inset. Third, they vary massively in contour length, with the thinner fibrils being significantly shorter at 24 h incubation, although the contour lengths clearly evolve directly with incubation time. The persistence length of the type III assembly was thus determined at longer incubation times, together with an assessment of the kinetics of fibrillation, in the presence and absence of salt. The type I fibrils, which are the most rigid ones, appear around 6 h of incubation, and grow up to ∼10 μm at 24 h. They are reminiscent of the typical amyloid-like structure from fibril-forming proteins.20,26 Figure 2a shows a progressive decrease in their contour length with heating time above 24 h and a narrowing of their length distribution. This shortening is due to breaking of the already formed long fibrils, because of continuous magnetic agitation (see Figure S2 in Supporting Information). The type II fibrils already appear around 2 h, mainly as 500 nm long filaments, and are quite flexible, bending on a few hundreds of nanometers (Figure 1, inset). Above 4 h incubation, they are several micrometers long and mostly wavy, resembling lysozyme sinusoid-like fibrils,25 but the precise evolution of their average contour length could not be determined, due to their tendency to aggregate together, even at high dilution. Their scarcity at longer incubation times also suggests that they are proto-filaments, that is to say intermediate structures that assemble into thicker multistranded amyloid fibrils upon incubation. The type III fibrils form during the first hours, as point-like aggregates. They become oligomers above 6 h (Figure 1) and grow with time into very flexible fibrils, up to a micrometer long (Figure 2b), that bend over few tens of nanometers (Figure 1, inset). Their contour lengths exceed those given in literature for such flexible ovalbumin fibrils.14−16 They have a typical worm-like morphology, similar to the HypF-N or α-lactalbumin protofibrils.3,27 What could first be interpreted as a “beadedlike” structure,11 built by small monomeric units of ∼25 nm in diameter (Figure 2b, 211 h zoom inset picture), is in fact resolved with ultrasharp AFM probe to most likely be lefthanded, partially twisted fibrils (Figure S3). Addition of salt to ovalbumin protein solution, prior to incubation at low pH and high temperature, has been shown to
Figure 2. Contour length evolution profiles with incubation time of (a) type I fibrils and (b) type III fibrils. The AFM height inset images have scale bars corresponding to 1 μm, except for the zoomed 211 h picture in (b) having a 500 nm width.
induce an increase in the fibrils contour length and lead to clustering at even greater NaCl concentrations.14 We are here interested in comparing the fibrillation pathways between low ionic strength and 50 mM NaCl conditions, having a closer look at the type of fibrils formed under these two conditions and at their growth kinetics. In this experiment, where only the ionic strength is varied, the type III fibrils are predominantly formed. Only a few type I and II fibrils can be found at 50 mM NaCl. However, although these fibrillar structures are similar to the ones identified in the low ionic strength conditions, the self-assembly pathway is slightly different. First, the oligomers develop to type III fibrils at shorter incubation times. At 9 h heating, when the nonsalted thin assemblies still resemble short oligomers, the 50 mM NaCl fibrils already grew to hundreds of nanometers in contour length (Figure 3a,b). Second, they continue to grow faster, eventually reaching (around 170 h) contour lengths 1 order of magnitude greater than usually reported in literature. At longer times, in the salted conditions, the long (≥3 μm) and flexible (Lp ≈ 63 ± 7 nm) type III fibrils start to form gel-like networks, which no longer allow for precise contour length estimation by AFM. In Figure 3b, it appears clear that 170 h of incubation are required for the low ionic strength thin fibrils to reach contour lengths as long as the ones observed after only 24 h in the salted conditions. 4215
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type I fibrils (Figure 4a,b) present a periodic left-handed twist (pitch ∼110 nm; distance from one height maxima to the next ∼55 nm). It suggests the presence of two subpopulations among type I fibrils, one of them being multistranded twisted ribbons, although these two subpopulations do possess nearly identical maximum heights. More rarely, flat ribbon-like larger lateral assembly can occur, typically ∼100 nm large and 6 nm thick (Figure 4c), consistent with observations recently made for hen egg white lysozyme and β-lactoglobulin proteins fibrils.21
Figure 3. Contour length evolution profiles with incubation time of (a) type III fibrils in 50 mM NaCl solution and (b) type III fibrils with (red) or without (black) salt, with (triangle) or without (square) oligomers (aspect ratio = 1) taken into account in the statistics. Error bars represent the average deviation. The inset pictures are AFM height images with scale bars corresponding to 1 μm.
Third, the type I and II aggregates are almost nonexistent in the presence of 50 mM NaCl. The type I rod-like fibrils are seen above 3 h, but mainly as short fragments. No long fibrils, as in 24 h no salt (Figures 1 and 2a), form in this experiment. Some type II fibrils are identified between 9 and 24 h of incubation, but to a much lower extent than in the low ionic strength conditions. Salt addition before starting the incubation has, thus, a significant impact on the fibrillation kinetics and on the types of fibrils formed. It has already been shown that the pH plays an important role in the type of fibril formed and is decisive for amyloid-like aggregation versus amorphous assembly.19,28,29 It has also been pointed out that, by modifying the protein net charge, as is done with engineered ovalbumin variants, the morphology of self-assemblies was varied. Increasing the ovalbumin net charge indeed leads to stiffer fibril formation at pH 7.30 One can thus conclude that the screening of the electrostatic repulsions between the fibrils building blocks, through addition of salt, favors the formation of the thinner and more flexible type III structures rather than the stiffer type I fibrils. It remains to be determined if the type III kind of fibrils have a different internal structure compared to type I, which will be discussed later in this manuscript. Besides the three main types of fibrils described above, additional fibrillar morphologies were observed. Some of the
Figure 4. AFM height image of (a) a periodic twisted ovalbumin fibril, (b) its profile along the white arrow, (c) a multistranded flat ribbon, and (d) a left-handed twisting ovalbumin fibril formed by thin flexible filaments of type III in the salted conditions. The scale bars represent 500 nm.
Concerning type III fibrils, around 265 h heating, the lefthanded worm-like thin protofilaments tend to aggregate together and seem to assemble into stiffer periodically twisted left-handed ribbons (Figure S4). This additional self-assembly mechanism, here commonly appearing at long incubation times, occurs in a similar way in the salted experiment after 170 h incubation, as shown in Figure 4d. In the early stage of the 4216
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Figure 5. DLS CONTIN analysis plots of hydrodynamic size distribution evolution with heating time for (a) the ovalbumin fibrils solutions containing all types of fibrils and (b) the same solutions containing only the type III fibrils after filtration. SAXS intensity versus scattering vector curves are plotted in a log−log scale for the type III unsalted fibrils obtained after (c) 170 h and (d) 265 h heating. The best fitting parameters (from the theoretical fits in black dashed lines) of the flexible cylinder model with polydisperse length are displayed on the graphics.
fibrils corresponds well to the left peak of the bimodal distribution shown for the nonfiltered solution in Figure 5a. To get additional insight into the type III self-assembly characteristics, SAXS experiments were run on the filtered solutions at different times. In Figure 5c, the double logarithmic representation of the scattering intensity versus scattering vector q is plotted for the flexible worm-like fibrils (unsalted) at 170 h incubation. The scattering curve is fitted by the flexible cylinder model with polydisperse contour length (Supporting Information). The best fitting parameters are displayed on the figure. The radius of the fibrils and, hence, the diameter, extracted from this fit, are larger than the average height of the type III fibrils determined by AFM (Figure 1). This difference can be explained by (i) the larger diameter of a fully hydrated fibril in solution compared to its height when deposited on a mica substrate and imaged in the dry state, (ii) the residual short fragments of the type I thick fibrils left in solution after filtration together with the type III ones, and (iii) the interval of confidence of the fitting parameters. The polydispersity, Kuhn length, and contour length estimated by the theoretical fitting are in good agreement with the respective polydispersity in contour length, persistence length, and average contour length, which are determined by AFM for the thin fibrils at 170 h. At 265 h, in Figure 5d, the increase in the contour length estimated by the fit is also very consistent with the previous AFM analysis. The hydrodynamic radii (Rh), obtained at the different heating times, are compared in the Table 1 with the radii of gyration (Rg) measured by SLS on the same filtered type III fibrils. The shape factor, ρ = Rg/Rh, lies in the range of the
self-assembly process, the twist can nevertheless be irregular (not yet perfectly periodic). To further characterize the fibrils behavior in solution, bulk experiments such as static and dynamic light scattering were performed. The results of DLS experiments, made on nonsalted fibrils at different times, are plotted in Figure 5a. The bimodal distributions, arising from fitting of the different crosscorrelation decay times, correspond to the two main types of self-assembly identified by AFM: types I and III. The thin fibrils, being in the oligomeric state at 24 h, show hydrodynamic radii (Rh) around 14 nm. The second peak, whose distribution is mainly centered around 200 nm at this time, is representative of the thicker type I fibrils. With increasing incubation time, the left peak shifts to higher Rh values, consistent with the previous observation of progressive growth of the type III fibrils (Figure 2b). The slight decrease in Rh for the DLS right peak in Figure 5a, between 24 and 50 h, is consistent with the decrease in contour length of the type I fibrils upon agitation. Its subsequent increase, above 50 h, does not give further evidence of the expected breaking of those fibrils, as depicted in Figure 2a. This phenomenon can be explained by the formation of large aggregates around type I fibril fragments at long heating times, as pictured in Figure 5a,b, inset. From this AFM height image, one can notice that, at 265 h, long type III fibrils cluster around shorter and thicker rodlike type I fragments, thus, forming larger aggregates. In Figure 5b, the same DLS experiment was run, but this time on the filtered solution, which contains only the type III fibrils, formed at low ionic strength. The Rh evolution of these isolated thin 4217
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sequences. Additionally, a difference in intrinsic stiffness, such as the one underlined here, could also be due to a considerable variation in the secondary structure. We, hence, investigated the two options, using SDS-PAGE and MALDI-MS on the one hand and CD, FTIR, and WAXS on the other hand.
Table 1. Type III Fibrils Hydrodynamic Radius, Radius of Gyration, and Contour Length Evolution with Heating Time Determined by Light Scattering and AFM time (h)
DLS Rh (nm)
SLS Rg (nm)
AFM Cl (nm)
24 50 100 170
14 22 27 35
22 41 49 56
20 ± 13 34 ± 26 65 ± 56 116 ± 90
values expected for a linear polymer chain in a good solvent. The predicted value of ρ = 1.6 for a linear monodisperse polymer in a good solvent,31 can increase above 1.7 when taking into account the polydispersity of the chains, consistent with the shape factors determined for the type III ovalbumin fibrils.32 These experimental radius values are compared to the theoretical Rg and Rh, based on the AFM contour length data (Supporting Information). We have recently reported that peak force QNM AFM allows for high-resolution measurement of the intrinsic properties of amyloid fibrils.33,34 This technique gives access to structural and nanomechanical properties of single fibrils deposited on a substrate. In Figure 6a,b, the height and Young's modulus images show together that the two main types of fibrils, I and III, have different values of Young's moduli. Figure 6c gives further details about the modulus values distribution (statistics made on more than 500 fibrils each). The type I fibrils (Figure 6 dark gray arrow and distribution) have a modulus of about 2.8 GPa, which is consistent with the values found for α-synuclein, insulin, and other amyloid-like fibrils, typically in the range of 2−4 GPa.33,35 The type III fibrils (Figure 6, light gray arrow and distribution) have significantly, almost 3-fold, lower modulus values: around 1.1 GPa. It has been shown that morphologically different amyloid structures of the same peptide, which have different rigidity, can have the same Young's modulus.33 In our case, however, the main kinds of ovalbumin fibrils, types I and III, do not only have morphological or overall rigidity discrepancies, but also fully different intrinsic stiffness. This suggests a distinct constitution of those fibrils. The type III fibrils are moreover below the Young’s modulus range usually attributed to amyloid fibrils, suggesting an alternative type of assembly. A more amorphous or less-structured aggregation than β-sheets assembly36 could typically look like the flexible type III fibrils formed by ovalbumin here and could explain the significant difference in the Young's moduli. One can, thus, suppose that the two types of fibrillar aggregates are built from different ovalbumin peptidic
Figure 7. (a) SDS-PAGE results of ovalbumin solution at different heating times in hours. (b) MALDI-MS results of the type I fibrils formed at 24 h.
The SDS-PAGE results, in Figure 7a, prove that hydrolyzed ovalbumin fragments are present in solution after heat treatment. The native protein is completely fragmented after 20 h heating at pH 2 and 90 °C (Figure S6). Further hydrolysis takes place up to about 80 h, leading to the three main shortest bands below 7 kDa, which are still present at 265 h. It has recently been shown that the kinetics of hydrolysis plays an important role in the formation of different types of fibrillar morphologies.21 Here we observe that the amyloid-like type I fibrils are formed in the early stages of the incubation process at
Figure 6. AFM images of ovalbumin fibrils (a) height and (b) Young's modulus determined by PF-QNM. (c) Modulus distribution profiles for the type III fibrils (light gray arrow/distribution) and type I fibrils (dark gray arrow/distribution). 4218
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Figure 8. (a) CD results of type I fibrils at 24 h (24 h ret) and 170 h (170 h ret), type III oligomers and fragments in solution (24 h filt), fragments in solution at 170 h (170 h filt), and type III fibrils (170 h int). The inset table gives the CDPRO secondary structure proportions of the samples marked by a continuous line. (b) WAXS log−linear intensity plot of the type III (gray) and I (black) fibril powders. (c) ATR-FTIR absorbance curves in the amide I region for the ovalbumin (dashed) and the type III (gray) and I (black) fibrils.
fragments identified by MALDI-MS (Figure S8). One interesting fact to notice is that the larger fragment (m/z = 3877.9) has a significant peak area in the filtrate (Figure S8b), corresponding to the thin flexible fibrils or free fragments in solution, and is insignificant in the retentate, namely, the type I fibrils (Figure S8a). The same observation can be made in Figure S9a,b, where the thin type III fibrils also display this larger fragment, which could therefore be attributed to this type of structure, because it is not present in the free fragments remaining in solution (Figure S9c). These results provide strong evidence that the peptide sequence with the largest mass/charge ratio is only contained in type III fibrils and are consistent with the possibility that aggregation in type III fibrils could not be driven by β-sheet stacking, but rather by a more fractal-like colloidal aggregation. We thus showed that the two types of fibrils, the thin (III) and the thick (I), are not formed from exactly the same fragments, with a propensity of type III fibrils to include peptide sequences with large m/z ratio. A precise determination of all the fragments involved was, however, not possible due to the high number of AA sequences involved. Knowing that a difference could be found at the peptidic level, we further investigated if a variation in the secondary structure also exists between these two classes of ovalbumin fibrils. We thus separated the two types of fibrils in solution by ultracentrifugal filtration to compare their bulk conformation. In Figure 8a, circular dichroism results are presented for the different fractions, at two different incubation times. The black continuous line characterizes the dialyzed, freeze-dried initial ovalbumin protein in solution at pH 2, called “monomer”. The continuous red line, representing the long type I fibrils formed after a 24 h heat treatment, clearly shows a single minimum at 215 nm, characteristic of well-defined β-sheet structure. The same isolated fibrils after a 170 h incubation show a very similar curve (green continuous line). The CDPRO software results for α-helix and β-sheet content determination of the type I fibrils compared to the ovalbumin “monomer” are depicted in Figure 8a, inset table. Upon incubation, the protein loses αhelix content and gains β-sheet, supporting the amyloid nature of the semiflexible fibrils. The decrease in α-helix content at 222 nm, (Figure S10) is also apparent when considering the overall sample (nonfiltered solution). The helix content remains slightly higher in the salted case (e.g., more negative molecular ellipticity), where mostly type III fibrils are formed. In Figure 8a, the molar ellipticity of the filtrates (dash-dotted lines), for example, the unconverted fragments remaining in solution at 24
low ionic strength. We also know that in the salted case, these long semiflexible fibrils do not form and that only few thick short fragments are found. Nevertheless, by comparing the hydrolysis evolution between the two experiments, only minor differences appear. The fragmentation of the ovalbumin seems to happen slightly faster in the nonsalted sample (Figure S6), the band around 25 kDa remaining intense for longer time in the salted case, which is consistent with presence of type I fibrils in unsalted samples, assuming that these fibrils are composed of short peptidic fragments. However, the shielding of electrostatic repulsions, through addition of salt, remains a more plausible explanation for promoting an easier and faster type of aggregation into type III fibrils. At long incubation times, where the same sizes of fragments are present in solution, the types of fibrils observed are similar in both cases: mainly long thin type III fibrils are present, with some thick periodic fibrils that seem to be built from the thin ones. Knowing that short peptidic fragments are key components of ovalbumin fibrils, we intended to determine, by MALDI-MS, the exact amino acid (AA) sequences, from the pristine protein,37 involved in both types of fibrils (types I and III). In Figure 7b, several peaks, corresponding to the constitutive fragments of the isolated type I fibrils at 24 h, are identified. The most probable AA sequences are researched for several main peaks. The Mascot search results gave individual ion scores, high enough to indicate identity or extensive homology, in the case of the two AA sequences marked in Figure 7b. The two peaks correspond to the same sequence Ser168-Lys189, with only one additional Aspartic acid residue for m/z = 2595.4 (Figure S7). Interestingly, the sequence SQTAMVLVNAIVFKGLWEKAFK is mainly composed by a fragment that is in the β-strand conformation in the native ovalbumin protein: TAMVLVNAIVFKGLWEK. Several algorithms, such as TANGO, used for the prediction of the proteins core regions for amyloid fibrils formation, moreover, designate the MVLVNAIVFK sequence for its high β-aggregation propensity. This last peptide, when obtained by a synthetic approach, has been shown to form amyloid fibrils in HFIP, Tris-HCl buffer with NaCl, after heat treatment.38 In accordance with this previous work, we additionally prove here that such a fragment is included in ovalbumin amyloid fibrils, which are simply formed by heat treatment of the native protein at pH 2. This reinforces the idea that hydrolysis and fragmentation of the native protein allows for amyloid-like aggregation. We also intend to shed light on the discrepancies between type I and III fibrils. Differences can be seen in the constitutive 4219
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amyloid fibrils of class I have a Young's modulus of 2.8 GPa, while the thin flexible fibrils of class III have a modulus around 1.1 GPa, well below the typical Young's modulus range reported for amyloid fibrils.
or 170 h, obviously have a random coil profile, with minima between 195 and 200 nm. At 170 h, the type III worm-like fibrils are long enough to be separated from the filtrate (170 h filt) and can be measured alone, that is, without nonaggregated fragments, as an intermediate isolated fraction (170 h int). They show a low ellipticity signal (dashed green line). No predominant structure can be extracted: the thin type III fibrils seem to contain some α-helix, β-sheet, and random coil but no deep peak of molar ellipticity emerges. There is, thus, no signature of a really well-defined specific secondary structure. This marks an evident and important difference in the structure between the type I and III fibrils. Those two classes of aggregates, after drying, could also be compared by WAXS (Figure 8b). The type I semiflexible fibrils, showing high βsheet content in CD, also shows the characteristic peak of 4.7 Å d-spacing in the WAXS signal, corresponding to an axial reflection of the inter strand spacing in the cross-β structure typical of amyloids.39 The second broader peak, around 10 Å, also corresponds to the expected equatorial reflection between 8 and 12 Å, accounting for the spacing between two or several β-sheets stacked in the fibril. In contrast, the type III fibrils rather show a random type of signal, with no sharp peak but a broad distribution, confirming the CD results. Scattering around 10 Å, typical of β-sheet stacking, is nonetheless still detectable in the type III fibrils, pointing at some possible ordering at this length scale. FTIR results (Figure 8c) further confirm that the two types of fibrils have a structure that differ from the initial ovalbumin conformation and that are significantly disparate among each other. The wavenumber range between 1647 and 1662 cm−1, characteristic of α-helical content, is greater, relative to the absorbance at the other wave numbers, in the case of the type III fibrils. Moreover, the crossβ region, characteristic of amyloid structures, between 1611 and 1630 cm−1, is much more predominant in the type I fibrils. The additional deconvolution details of the three curves are plotted in Figure S11. The combination of these last three experiments (Figure 8) thus prove that the type III worm-like fibrils, even if containing some β-sheet secondary structure, are significantly more “amorphous” types of aggregates than the ovalbumin type I amyloid-like fibrils. Such a difference in secondary structure, associated with two different fibril flexibilities, is comparable to the observations made on β-lactoglobulin fibrils morphology, which depends on protein preferential aggregation (between flexible and amyloid-like semiflexible fibrils) while varying the protein initial concentration.36
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ASSOCIATED CONTENT
S Supporting Information *
Supplementary figures: AFM height images, SDS-PAGE, MALDI-MS, and FTIR results. Comparison of the theoretical and experimental Rg and Rh. SAXS fitting model. 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: raff
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors would like to thank Ivan Usov for his kind contribution to the improvement of the fibrils tracking and characterization algorithms. The MALDI-MS experiments were done at the Functional Genomics Center in Zürich by Dr. Serge Chesnov, who also provided helpful advices.
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CONCLUSIONS We have demonstrated that ovalbumin can form several types of fibrils at pH 2 and 90 °C. Two main morphologies were characterized in this work. First, the thin and flexible worm-like fibrils, type III, which were reported to form in previous works, are here shown to grow up to lengths of micrometers and be prone to aggregation into thicker periodically twisted ribbons. Second, thicker and more rigid (semiflexible) type I fibrils, which showed typical amyloid characteristics, were developing preferentially at low ionic strength. The secondary structure of type I and III fibrils were investigated in detail: CD, WAXS, and FTIR results together prove that the semiflexible type I fibrils are β-sheet rich amyloid fibrils, whereas the type III fibrils do not have the typical amyloid fingerprint, as they do not possess such a specific ordered secondary structure. This difference can also be quantified in terms of the fibrils intrinsic stiffness: the 4220
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