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Functional ethanol-induced fibrils: Influence of solvents and temperature on amyloid-like aggregation of beta-lactoglobulin
Journal of Food Engineering 270 (2020) 109764
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Functional ethanol-induced fibrils: Influence of solvents and temperature on amyloid-like aggregation of beta-lactoglobulin Jil J. Kayser a, Philipp Arnold b, Anja Steffen-Heins a, Karin Schwarz a, Julia K. Keppler a, c, * a
Institute of Human Nutrition and Food Science, Division of Food Technology, Kiel University, 24118 Kiel, Germany Anatomical Institute, Kiel University, 24118 Kiel, Germany c Laboratory of Food Process Engineering, Wageningen University, Bornse Weilanden 9, 6708WG, Wageningen, P.O. Box 17, 6700 AA, Wageningen, the Netherlands b
A R T I C L E I N F O
A B S T R A C T
Keywords: Functional fibrils Lactoglobulin Whey protein Amyloid aggregation Ethanol Solvent-induced protein denaturation
Solvent-induced fibrillar aggregates of beta-lactoglobulin occur only in a certain balance between hydrophobic forces and electrostatic interactions. We hypothesize, that different hydrophobic solvent molecules as well as rising temperatures influence this equilibrium and thus the optimum to produce amyloid aggregates. Dimethyl sulfoxide (DMSO), methanol and ethanol all resulted in polydisperse solutions with worm-like and spherical-aggregates, albeit to different degrees: the volume fraction required for aggregation was DMSO (50%) > methanol (40%) > ethanol (30%) which does not reflect their hydrophobicity. Further solvent addition decreased the fibrillar aggregation again. Increasing the temperature by 10–20 K decreased the solvent con centration needed to induce amyloid-like aggregates. A targeted production of solvent-based amyloid-like aggregates is therefore not only dependent on the hy drophobicity of the solvents, but also on their direct interaction with the protein (denaturation).
1. Introduction Among temperature-induced protein aggregates, amyloid aggregates are characterized by their stacked beta-sheet conformation, which is formed by several individual self-associating building blocks (Kroes- Nijboer et al., 2012), mainly driven by hydrophobic interactions and further stabilized by hydrogen bonds (Nicolai et al., 2011). Different types of temperature-induced functional amyloid and amyloid-like ag gregates from whey protein beta-lactoglobulin (BLG) can be distin guished by their morphology, depending on pH value, ionic strength, stirring speed, temperature, incubation time and protein concentration (Cao and Mezzenga, 2019; Keppler et al., 2019; Heyn et al., 2019; Serfert et al., 2014; Nicolai and Durand, 2013; Loveday et al., 2009; Jung and Mezzenga, 2010). Amyloid aggregates of different morphologies have increasingly become the focus of research in recent years owing to interesting functional properties that make them suitable as e.g. thick eners, gelling agents, stabilizers in emulsions and foams as well as for microencapsulation, film formation and surface coatings (Cao et al., 2019; Wei et al., 2017; Serfert et al., 2014; Humblet-Hua et al., 2013; Kroes-Nijboer et al., 2012; Li et al., 2012; Isa et al., 2011; Knowles et al.,
2010; Loveday et al., 2009; Akkermans et al., 2008a). However, incu bation during 5 h at temperatures >80 � C also results in oxidative pro tein deterioration (Keppler et al., 2019) and high production costs. Apart from temperature induced protein denaturation, other sub stances such as urea and guanidine hydrochloride as well as molecular crowding agents such as polyethylene glycol can also induce ThioflavinT reactive fibrillar aggregates (Ma et al., 2013; Akkermans et al., 2008b; Hamada and Dobson, 2002). Their assembly mechanism and structure may differ from temperature-induced amyloids observed at pH 2, which is why they are often called “amyloid-like” fibrillar assemblies. In addition, various alcohols and solvents were found to unfold proteins and stabilize the peptide structure by lowering the polarity of the solvent and binding to the protein. The alcohol-induced denaturation of the native protein is usually followed by the induction of a α-helix structure and further transformation into a predominant β-sheet conformation (Hirota-Nakaoka and Goto, 1999; Hong et al., 1999; Yoshida et al., 2012). At high protein concentrations, this mechanism can be followed by amyloid-like aggregation, which is caused by interactions between the protein molecules (Yoshida et al., 2012). Such amyloids induced by food additive ethanol could be an alternative to the
* Corresponding author.Laboratory of Food Process Engineering, Wageningen University, the Netherlands Bornse Weilanden 9, 6708WG, Wageningen. P.O. Box 17, 6700 AA, Wageningen. E-mail addresses: [email protected] (J.J. Kayser), [email protected] (P. Arnold), [email protected] (A. Steffen-Heins), info@foodtech. uni-kiel.de (K. Schwarz), [email protected] (J.K. Keppler). https://doi.org/10.1016/j.jfoodeng.2019.109764 Received 4 September 2019; Received in revised form 11 October 2019; Accepted 13 October 2019 Available online 16 October 2019 0260-8774/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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Journal of Food Engineering 270 (2020) 109764
temperature-induced production of functional amyloids from BLG. To better understand the alcohol and solvent-induced amyloid-like aggregation, the influence of different solvents on the model protein BLG has been investigated in the past. Alcohols that have already been studied more intensively in combination with the protein are 2,2,2-tri fluoroethanol, 3,3,30 ,30 ,30 -hexalfluoro-2-propanol, methanol, ethanol and propane-2-ol with varying influencing factors such as protein con centration, pH value, temperature, alcohol concentration and incuba tion time (Hong et al., 1999; Gosal et al. 2002, 2004; Hirota-Nakaoka and Goto, 1999; Yoshida et al. 2012, 2014; Uversky et al., 1997; Mousavi et al., 2008; Maity et al., 2016). To the best of our knowledge the effect of ethanol on BLG amyloid aggregation at an acidic pH of 2.0 was investigated only at room temperature (Gosal et al., 2004; Uversky et al., 1997) while only at a pH of 7.0 a variation of temperature (25–70 � C) was included (Yoshida et al., 2014). So far, no study specif ically analyzed the effects of ethanol on BLG under similar conditions as applied for the preparation of functional temperature-induced amyloids. To investigate the functionality for the targeted production of func tional ethanol-induced BLG amyloids in the future, however, a deeper understanding of the effect of the food additive ethanol on protein ag gregation is required. For better comparability, the aggregation behavior of BLG induced by this alcohol was investigated in an envi ronment similar to the one used for temperature-induced functional amyloid preparation (i.e., 2.5% protein, pH 2, low ionic strength, 5 h incubation and gentle stirring at 350 rpm), albeit at lower temperatures between 30 � C and 50 � C. To get a better understanding of the under lying mechanisms, two further non-food grade solvents were added, one with a higher (methanol) and one with a lower polarity (DMSO) than ethanol. Similar methods typically used to study functional fibrils, such as beta-sheet stacking by Thioflavin-T test (ThT), conformation by FTIR, amyloid building blocks by size-exclusion chromatography (SEC), and morphology by TEM, are used in the experimental setup. The experiments should identify suitable combinations of incubation time, solvent concentration and temperature for the targeted production of solvent-based fibrillar aggregates that can be used in the future to keep production costs low and avoid oxidative protein damage.
2.3. Determination of amyloid aggregation 2.3.1. Thioflavin-T assay The analysis was performed according to the protocols of Loveday et al., (2012), Akkermans et al., (2008c) and Serfert et al., (2014) using a fluorescence spectrophotometer (Cary Eclipse, Varian GmbH Darmstadt, Germany). A stock solution of ThT in phosphate-NaCl buffer (10 mM phosphate and 150 mM NaCl, pH 7.0) was filtered through a 2.0 μm pore size membrane filter (Porafil, Macherey-Nagel GmbH & Co. KG, Düren, Germany) and stored in a dark bottle protected from light. The samples taken were diluted to 1 wt% with demineralized water set to pH 2.0. 48 μl of the diluted protein solution were pipetted into 4 ml of the ThT stock solution. This mixture was immediately vortexed for 20 s and measured after one minute of incubation at 440 nm excitation and 482 nm emission. For the measurement, the 1 � 1 cm quartz cu vettes with four polished sides were used. 2.3.2. Fourier-transform-infrared-spectroscopy (FT-IR) ATR-Fourier transform infrared spectroscopy (FT-IR) was performed on a Confocheck™ Tensor 2 system (Bruker Optics, Ettingen, Germany) optimized for protein analytics in solutions and a thermally controlled BioATR2 unit as described before (Keppler et al., 2019). The samples were first diluted to 1 wt% with demineralized water at pH 2.0 and the respective volume percentage solvent added. Measure ments were conducted at a temperature of 25 � C against the respective solvent mixtures without protein as background and averaged over 120 scans at a resolution of 0.7 cm 1. For evaluation, the measured spectra in the frequency range of the amide band І (1590 - 1700 cm 1) under went atmospheric correction and were then vector-normalised. The second derivative was calculated using 9 smoothing points. 2.3.3. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was performed as described before (Arnold et al., 2014). In brief 5 μl of sample were loaded onto previously negative glow discharged continuous carbon grids (Science Service, Munich, Germany). After adsorption of the sample for 10 s, access sample was blotted off and the grid was stained with a half-saturated solution of uranyl acetate. After air drying the sample was transferred into an JEOL 1400 Plus electron microscope operating at 100 kV (JEOL, Munich, Germany). Images were acquired utilizing a F416 4kx4k digital camera (TVIPS, Munich, Germany).
2. Materials and methods 2.1. Materials Beta-Lactoglobulin (BLG) was obtained from Davisco Foods Inter national, (Inc., Eden Prairie, US) with 97% protein and 96% BLG in dry matter. Thioflavin-T (ThT >95%) was obtained from EMD Chemicals Inc.. Guanidine hydrochloride (GHCl �99.5%) was obtained from Carl Roth GmbH þ Co. KG and 1,4-dithiothreitol (DTT �99,0%) originated from Sigma Aldrich Inc. All other chemicals were of analytical grade and obtained from Sigma Aldrich Inc.
2.3.4. Size exclusion chromatography (SEC) An Agilent 1100 HPLC system equipped with a diode array detector set at 205 nm, 214 nm, 278 nm and 280 nm wavelength was used with a Superdex™ 200 increase 10/300 GL SEC column from GE healthcare (Bio-Sciences AB, Uppsala, Sweden). The samples were prepared as described in Keppler et al. (2019): 0.08 ml of the 2.5 wt% sample solution were added to 0.42 ml dithio threitol (DTT) and guanidine hydrochloride (GHCl) solution (100 mM Dithiothreitol and 8 M Guanidine dissolved in 0.15 M Tris HCl buffer (pH 8) and incubated for 60 min at room temperature under continuous shaking at 140 RPM. 0.215 ml of the 0.15 M Tris HCl buffer were added and the solution was vortexed again. The samples were then filtered through a 0.2 μl cellulose filter and 50 μl were injected in the column with 0.15 M Tris HCl buffer as eluent. The flow rate was 0.3 ml/min. The calibration kit “Gel Filtration Calibration Kit LMW” from GE Healthcare was used for calibration in similar conditions.
2.2. Preparation of BLG solutions and production of fibrils BLG was dissolved at room temperature in demineralized water. Then, a final volume of 100 ml was adjusted with demineralized solventwater so that the solution had a total protein concentration of 2.5 wt% BLG. The pH value of the solution was set to pH 2.0 with 6 M and 1 M HCl and the final solvent volume (ethanol, methanol or DMSO) was either 20%, 30%, 40% or 50%. After preparation, all samples were directly transferred to the incubation chamber based: 40 ml of the sample were placed in a Schott glass with closed screw caps (125 ml max volume) and heated for 5 h at 30 � C, 40 � C or 50 � C with constant stirring at 350 rpm using a water proof magnetic stirrer (2mag MIXdrive 6HT, 2mag AG, Munich, Germany). Every 30 min, a sample of 1 ml was taken, cooled in an ice-water bath and stored at 4 � C until further use.
2.3.5. Electron paramagnetic spectroscopy (EPR) The micro polarity of the various solvent mixtures, in concentration series of 10–70 wt% alcohol were determined by the electron para magnetic resonance spectroscopy (Bruker Elexsys II E500 spectrometer, Rheinstetten, Germany) using the spin probe Tempol0.5 mM Tempol (4 Hydroxy Tempo 97%; Sigma Aldrich) and the percentage of the solvent were added to the sample and the sample was adjusted to a pH value of 2
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Journal of Food Engineering 270 (2020) 109764
2.0 with 1 M and 0.5 M HCl. The samples were measured three times. The following parameters were used for the spectra acquisition: micro wave frequency, 9.85 GHz; modulation frequency, 100 kHz; microwave power, 0.2 mW; modulation amplitude, 1.30 G; time constant, 10.24 m s; conversion time, 10.3 m s. The center field was optimized to 3511 G with a sweep width of 60 G.
(Ban et al., 2003; Xue et al., 2017). The reference sample (protein so lution without alcohol addition) as well as the one with 20% ethanol showed a constant low ThT value of ̴ 4 Int. A.U. during 5 h incubation (Fig. 2 A). However, an exchange of 30% ethanol resulted in a strong fluores cence increase already in the first 90 min (~10–70 Int. A.U.) and reached a final maximum after 5 h of 80 Int. A.U. Similar findings were evident for higher ethanol volumes, although the maximum ThT fluo rescence derived after 5 h incubation decreased with further ethanol replacement (i.e., 80 Int A.U., 60 Int A.U. and 50 Int A.U. fluorescence maximum for 30%, 40% and 50% volume replacement). A similar effect was observed for methanol (Fig. 2 B), although here the ThT-fluorescence increased significantly only with more than 30% methanol and for DMSO (Fig. 2 C), the ThT-fluorescence was only evident after more than 40% solvent replacement. The most ThT-active solvent mixtures (i.e., �30% ethanol, �40% methanol and �50% DMSO) showed a rapid increase of ThTfluorescence in all three solvents, indicating a temporal component in the solvent induced amyloid aggregation. However, methanol has the highest initial values at the beginning of incubation, whereas DMSO shows the slowest reaction.
2.3.6. Statistics Statistical analysis was conducted with the software GraphPad PRISM (version 8.2.1, GraphPad Software, San Diego, USA). A two-way ANOVA with Tukeys post hoc test was conducted at 0.05 level of significance. 3. Results 3.1. Effect of alcohol addition on solvent polarity at room temperature The nitroxide spin probe Tempol dissolved in hydro-ethanolic solu tion resulted in a typical three-line spectrum of a free tumbling nitroxide (Fig. 1). This is apparent in the same peak amplitude and peak width of all three lines as well as in the equal distance between the peaks, the socalled hyperfine splitting aN [G]. The hyperfine splitting parameter aN [G] given by the distance between the low field peak (hþ1) and the middle field peak (h0) is indicative for the micropolarity of the spin probe environment being lower in more non-polar environments. An increasing volume replacement by alcohol from 10 to 70% reduced aN, indicating a less polar environment. Table 2 lists the observed hyperfine splitting parameters for aqueous ethanolic, methanolic and DMSO so lutions at room temperature. The micropolarity can range between aN ¼ 17 [G] for an highly polar environment and aN<13 [G] for nonpolar solvents (Berliner, 1976). All solutions with 10%–30% solvent replacement were highly hy drophilic, as indicated by aN between 16.9 G and 16.8 G. Generally, the observed aN decreased with increasing volume replacement in the experimental range of 30%–50% in the order ethanol ¼ DMSO > meth anol. At 70% solvent replacement, a minimum aN of 16.27 G was observed for DMSO, while ethanol reached 16.37 G and methanol 16.48 G. This order confirms the relative polarity listed in the specifi cation (Table 1).
3.2.2. Infrared spectroscopy: increasing alcohol concentrations The influence of the solvents on the secondary structure of BLG was investigated by ATR-FT- IR before (0 h) and after incubation (5 h). The amid І region (1600-1700 cm 1) is indicative for conformational changes of the protein (Haris and Chapman, 1995; Nilsson, 2004). A typical native protein spectrum in the second derivation is shown in Fig. 3 A. Spectral band minima at 1635 cm 1 reflect intramolecular native beta-sheets, while ̴ 1655-1658 cm 1 is indicative for helical content and 1619-1620 cm 1 is often observed for intermolecular ag gregates typical for denatured and aggregated proteins and often observed in amyloid aggregates (Keppler et al., 2019; Heyn et al. 2019; Sarroukh et al., 2013; Matheus et al., 2006). In the control experimental approach with 0% volume replacement of solvent (Fig. 3A), the initially strong native β-sheet signal at 1635 cm 1 (black continuous line) did not change significantly during the 5 h incubation (black dotted line). With increasing ethanol replacement to 30%, the α-helix signal (1655-58 cm 1) increased directly after incubation at 0 h (Fig. 3 D), then decreased at the expense of a strong signal at 1619 cm 1 in the 5 h incubated samples, indicating a fast initial helix formation followed by intermolecular β-sheets typical for amyloids or aggregated protein which were not present at the beginning of the incubation (0 h). This signal was also present at 40% and 50% alcohol replacement although its intensity decreased from 30%
3.2. Influence of alcohol and temperature on amyloid aggregation 3.2.1. Thioflavin-T fluorescence: increasing alcohol concentration ThT fluorescence is frequently used for analysis of amyloid aggre gates because the ThT molecule binds tightly to assembled beta-sheets
Fig. 1. Exemplary electron paramagnetic resonance spectra of spin probe tempol with 10% or 70% hydro-ethanolic solution at room temperature. The line indicates the distance between the low and the middle field peaks. 3
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Table 1 Physico-chemical properties of the three solvents. Molar mass [g/mol] Density at 20 � C [g/cm3] 30% volume exchange [g/g water] 30% volume exchange [M/M water] Miscibility with water Relative polarity Boiling point [� C] Molecular structure
Ethanol
Methanol
DMSO
46.07 0.78–0.81 ~0.33–0.34 ~0.13 Fully miscible 0.654 78
32.04 0.79 ~0.35 ~0.2 Fully miscible 0.762 65
78.13 1.1 ~0.47 ~0.1 100 g/l 0.444 189
much higher length of about 100–150 nm.
Table 2 Hyperfine splitting parameters aN [G] for solvent-water mixtures at room tem perature. Values indicated by different superscript letters (a to c) are signifi cantly different in a row, p 0.05. All measurements were conducted in triplicate and listed as mean and standard deviation. Solvent (%)
Ethanol
Methanol
3.2.4. Size exclusion chromatography: increasing alcohol concentration The results confirm the presence of BLG monomers with a size of ̴ 19.1 kDa ( ̴ 51 min elution time according to calibration, monomeric BLG has a theoretical size of 18.4 kDa) and some aggregates ( ̴40 min elution time). These aggregates could be dimers. No clear evidence of protein hydrolysis during incubation can be observed (Fig. 5 A, B and C): Protein hydrolysates with a size smaller than monomeric BLG would have led to signals with higher elution times (>51 min).
DMSO
Micropolarity aN [G] 10 20 30 40 50 60 70
16.93 16.89 16.76 16.70 16.54 16.46 16.37
�0.00 a �0.03 a �0.00 b �0.06 a �0.03 b �0.06 b �0.03 b
16.91 16.86 16.84 16.76 16.64 16.60 16.48
�0.03 �0.03 �0.03 �0.00 �0.00 �0.03 �0.03
a a a a a a a
16.91 16.88 16.82 16.70 16.56 16.43 16.27
�0.03 a �0.00 a �0.00 a �0.06 a �0.03 b �0.03 b �0.03 c
3.3. Influence of temperature and alcohol on amyloid aggregation 3.3.1. Thioflavin-T fluorescence: increasing temperature In a second experimental approach, the influence of temperature on the aggregation behavior of BLG in solvent solutions were tested. For ethanol (Fig. 6 A), increasing the incubation temperature from 30 � C, to 40 � C, or 50 � C increased the aggregation speed significantly. A ThT fluorescence of 70 Int A.U. is reached in 1.5 h while it takes 1 h at 40 � C and 0.5 h at 50 � C. However, it seems that further incubation has a detrimental effect especially at 50 � C and the fluorescence decreases again to 60 Int. A.U. For methanol (Fig. 6 B) a similar effect can be observed although to a much stronger extent. Here, almost no ThT reactivity is evident at 30 � C, while it increases strongly at 40 � C and 50 � C. Again, further incubation to 5 h reduces the fluorescence slightly. For DMSO (Fig. 6 C) ThT fluorescence is also positively affected by increasing temperature, although here �50 � C are needed to obtain any significant signal.
to 40%–50% ethanol (Fig. 3 F). The native β-sheet signal decreased continuously after surpassing 20% ethanol (Fig. 3 E). For methanol (Fig. 3 B, D, E and F) a similar effect as for ethanol can be observed, although here, a first shift to the 1619 cm 1 waveband is only evident at 40% alcohol replacement and also only after 5 h incu bation. Likewise, the intensity of the intramolecular β-sheets decreased continuously with increasing methanol addition, the α-helical signal increased only initially (0 h) and to a lower extent than observed for ethanol, this effect was no longer evident after 5 h incubation. With DMSO (Fig. 3 C, D, E and F), a large volume exchange of 50% is necessary to induce intermolecular beta-sheets at 30 � C after 5 h incu bation. Below that, DMSO seems to have no strong effect on the helix or native β-sheet conformation, since 0 h and 5 h signals were not signifi cantly different except for a native β-sheet increase at 30% (Fig. 3 E). Only at a volume exchange of 50% DMSO noteworthy conforma tional changes occur such as the strong signal for intermolecular betasheets at 1619 cm 1.
3.3.2. Infrared spectroscopy: increasing temperature ATR-FTIR spectra confirm the findings of ThT. Between 30 � C and 50 � C a strong signal for the intermolecular beta-sheet aggregation at 1619 cm 1 is evident for ethanol (Fig. 7 A). For methanol this signal only occurs for temperatures similar or higher than 40 � C and for DMSO a first effect can be observed only at 50 � C.
3.2.3. Transmission electron microscopy: increasing alcohol concentration To confirm the presence of fibrillar protein aggregates, representa tive TEM images of the samples with 30% volume exchange by solvents were taken (Fig. 4A). Directly after solvent exchange (0 h) ethanol and DMSO samples show spherical aggregates of approximately 20–25 nm size. After 5 h incubation at 30 � C the TEM images of the sample give evidence that the morphology of the formed amyloid aggregates is short and worm-like with a length of ̴ 20–60 nm as well as partly radial. Similar aggregates can be observed in methanol, albeit significantly less pronounced. In DMSO only few aggregates are present, however, already at 0 h some spherical aggregates can be observed. In addition, also for a volume exchange with 40% methanol and 50% DMSO TEM images were taken (Fig. 4 B). After 5 h incubation at 30 � C the samples show also amyloid aggregates. A high proportion of amyloid aggregates can be observed in the sample with methanol, also with a short worm-like structure and a length of approximately 15–40 nm. In the solvent DMSO only a few amyloid aggregates are present but with a
4. Discussion 4.1. Influence of increasing volume exchange with solvents on amyloid aggregation 4.1.1. Maximum amyloid aggregation The results give evidence that all tested solvents influenced the amyloid-like self-association of BLG at pH 2 as determined by ThT (Fig. 2), FTIR (Fig. 3) and TEM (Fig. 4), albeit to a different extent: The alcohol or solvent volume fraction needed for inducing amyloidlike aggregation was in the order DMSO (50%) > methanol (40%) > ethanol (30%) (Fig. 4B). The individual concentration required to induce aggregation can be traced back to several factors: If the polarity of the two alcohols methanol and ethanol is compared 4
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Journal of Food Engineering 270 (2020) 109764
Fig. 2. Thioflavin-T fluorescence during 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol, B) Methanol und C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean and standard deviation of three samples.
(Table 2), it can be observed that a volume exchange of 30% ethanol results in the same polarity as a volume exchange of 40% methanol. A volume exchange of 50% ethanol and 50% DMSO, which have approximately the same ThT values after the end of the incubation period (Fig. 2), also shows a very similar polarity. A lower polarity of the solvent compared to water leads to a weakening of the hydrophobic interactions as well as to a strengthening of the electrostatic interactions (Hong et al., 1999; Hirota-Nakaoka and Goto, 1999; Nikolaidis et al., 2017). The altered equilibrium can result in a conformational change into the α-helix structure (Hirota-Nakaoka and Goto, 1999; Hong et al., 1999; Yoshida et al., 2012), which is also evident in FTIR (Fig. 3) where helix formation increased immediately after incubation at elevated solvent exchange for ethanol and methanol. Similar to the present findings, for ethanol it is known that a conformational transition to the α-helix structure takes place from a solvent concentration of 20% on wards (Nikolaidis et al., 2017). This helix transition is usually followed by a further transition to amyloid-like β-sheets at higher solvent concentrations (Yoshida et al., 2012). Interestingly, the ThT graphs (Fig. 2) show that in contrast to the rapid initial helix formation, the beta-sheet transition for 30% ethanol or 40% methanol takes approximately 1–2 h. This time difference between helix formation and beta-sheet transformation is also confirmed by the FTIR results, although only samples after 0 h and 5 h incubation were measured here (Fig. 3). Thus, the intermolecular β-sheet formation
initially follows slower kinetics than α-helix formation, although this effect is less pronounced with higher solvent addition, at least in the ThT test experiments. The stronger effect of ethanol than methanol on polarity can be traced back to the hydroxyl group having a negative effect on the described alcohol-induced effect. Thus the alcohol concentration required to induce the conformational transition decreases with increasing size of the alcohol molecule (Hirota-Nakaoka and Goto, 1999) (i.e., MW 46.06 for ethanol and 32.04 for methanol). In addition, some alcohols have a propensity to form micelles or clusters in aqueous solutions, which was found to further increase the observed effect on proteins (Hirota-Nakaoka and Goto, 1999). The organosulfur solvent DMSO has not been studied with respect to amyloid-like aggregation behavior of BLG and it acts differently than the alcohols. The sample with a volume exchange of 40% DMSO has the same polarity as the sample with 40% ethanol, but still a considerably lower effect on helix formation or amyloid-like aggregation was observed by ThT (Fig. 2 C) and FTIR (Fig. 3 C). Looking at the FT-IR spectra of the test series with DMSO (Fig. 3 C), it is evident that con trary to both alcohols, DMSO has no strong denaturing effect on BLG at low temperatures of 30 � C and low volume concentrations of up to 40%. Only from a volume exchange of 50% by DMSO, conformational tran sition can be observed. A preferential hydration of BLG was evident in the presence of low DMSO concentrations <50% volume exchange in 5
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Journal of Food Engineering 270 (2020) 109764
Fig. 3. FTIR second derivation spectra before and after 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol B) Methanol C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean of three individual measurements. A summary of D) the α-helix development, E) the native β-sheets and F) the intermolecular “amyloid” β -sheet development after 0 and 5 h incubation for each solvent and solvent concentration is given. The data are readouts of the above listed mean FTIR spectra and are thus only sketches without standard deviation to emphasize the temporal development.
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Fig. 4. TEM images of the respective samples directly after preparation (0 h) and after 5 h incubation at 30 � C A) with solvent solutions of 30% for ethanol, methanol and DMSO and B) solvent solutions with 40% for methanol and 50% for DMSO to confirm the presence of amyloid-like aggregates. Small inserts show a range of 200 nm.
B
400
0h 2h 5h
Intensity [A.U.]
Intensity [A.U.]
600
600
200
0
400
50
time [min]
60
0h 2h 5h
200
0 40
C 600
Intensity [A.U.]
A
40
50
time [min]
60
400
0h 2h 5h
200
0
40
50
time [min]
60
Fig. 5. Size exclusion chromatograms of dissociated BLG aggregates which were incubated in 30% solvent-water mixtures for 0 h, 2 h or 5 h at 30 C. A) ethanol, B) methanol and C) DMSO.
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Fig. 6. Thioflavin-T fluorescence during 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol, B) Methanol und C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean and standard deviation.
Fig. 7. FTIR second derivation spectra after 5 h incubation of beta-lactoglobulin with a solvent concentration of 30% (2.5% protein & 350 RPM). (A) ethanol (B) methanol (C) Dimethyl sulfoxide. The temperature was either 30 � C, 40 � C or 50 � C. All measurements are listed as mean of three individual measurements.
water, which resulted in preservation of the proteins native structure (Arakawa et al., 2007). In addition, because of the larger size (approx. 80 Da) DMSO is less able than ethanol to penetrate the native molecule in order to compete with non-polar small-range bonds. It is also evident from FTIR (Fig. 3) that intermolecular β-sheet formation of BLG is less pronounced in the presence of DMSO, which also confirms that there is less direct interaction between the hydrophobic groups of DMSO with those of the protein at low DMSO concentrations. However, >50% DMSO replacement finally has a denaturing effect on the protein. Only after this opening of the tertiary structure DMSO can interact and sta bilize the secondary conformation of BLG and lead to the observed larger amyloid-like aggregates formed in the presence of 50% solvent exchange with DMSO compared to ethanol and methanol (Arakawa et al., 2007; Inoue and Timasheff, 1972).
hydrophobic interactions initiate protein denaturation at lower tem perature. On the other hand, amyloid-like aggregates initially associate by hydrophobic forces and are then stabilized by hydrogen bonds. Therefore, it is likely that any further aggregation above a certain hy drophobicity (solvent concentration) is reduced, while the increasing hydrogen bond stability favors the stability of the formed fibrils. 4.2. Effect of temperature on solvent induced amyloid aggregation To alter the delicate balance between hydrophobic forces and hydrogen bonds further, the following experiments were conducted at a fixed solvent exchange of 30% but with increasing temperature. As ex pected, higher temperatures can lower the threshold for the solvent induced amyloid-like aggregation up to a certain point, where further temperature increase than again also has a detrimental effect (Figs. 6 and 7). It is well known that increasing temperature decreases the dielectric constant of the solution (Suryanarayana and Govindaswamy, 1961). In the previous section it could be shown that at a polarity induced by a volume exchange of 30% by ethanol and a temperature of 30 � C, con ditions are present which seem to promote the formation of amyloid-like aggregates. If the polarity of the medium in 30% methanol were still too high to induce fibrillar aggregates (Table 2), increasing the temperature from 30 � C to 40 � C could reduce the polarity of the medium and thus restore the balance between hydrophobic and electrostatic interactions favored by amyloid-like aggregates in a simplified model. A further in crease in temperature further alters the balance between hydrophobic and electrostatic interactions, so that the formation of fibrillar aggre gates is then again less favorable. Hence, this could be one possible
4.1.2. Decreasing amyloid aggregation with increasing solvents There seems to be an optimum solvent concentration to initiate amyloid-like aggregation here. As is evident in ThT (Fig. 2) and FTIR (Fig. 3), solvent concentrations above a certain optimum result in a decline of the amyloid-like aggregation effects (i.e., �40% for ethanol, �50% for methanol and not measured for DMSO), while the α-helix conformation increases linearly (Fig. 3D) only in ethanol. Yamaguchi and coworkers showed that the TFE-induced amyloid aggregation of a microglobulin peptide followed a similar bell shape as observed in the present manuscript for BLG, and thus reached an opti mum after which an alpha-helical-conformation dominated again. They argued that this was due to a sensitive balance between decreasing hy drophobic and increasing polar interactions with increasing solvent concentration (Yamaguchi et al., 2006). On the one hand, decreasing 8
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explanation of the observed effects. Elevated temperatures could also enhance the solvent-protein in teractions due to the altered solvent hydrophobicity as well as due to increased molecular mobility of water and solvent. In addition, the molecular flexibility of BLG improves the accessibility of its non-polar amino acids to the solvent molecules. This would result in an acceler ated exchange of surface bound water molecules by solvent molecules via short range hydrophobic interactions (Chong et al., 2015) and consequently exert an effect on the protein β-sheet formation (Yoshida et al., 2014). This would especially explain the enhanced aggregation observed with DMSO at 50 � C, where a strong conformational change is evident in FTIR with 30% solvent replacement (Fig. 7), while this is not observed at lower temperatures. There are also reports about a lowered denaturation temperature of proteins in the presence of solvents (Glu giarelli et al. 2012), while normally, the whey protein would be dena tured at temperatures of ̴ 70 � C (Singh et al., 2014; Keppler et al., 2014). According to the present findings, the following temperature in crease will lead to a maximum amyloid-like aggregation at 30% solvent exchange (at the given protein concentration and pH value): For methanol, the temperature would have to be increased by 20 K (from 20 to 40 � C). For DMSO a temperature increase of 40 K is probably required (from 20 to 60 � C). For ethanol it can be assumed that a temperature increase >10 K reduces the required solvent concentration to <30%.
4.4. Comparison with temperature-induced amyloid aggregates of BLG The ThT values of approximately 80 Int A.U. observed here (Fig. 2) are in the same range as described for temperature-induced amyloid aggregates, however, due to the polydispersity of the solvent induced aggregate (worm-like and spherical), the fibrillar material could not be clearly separated from non-amyloid material by centrifugal ultrafiltra tion (results not shown), so that no conversion rate can be calculated here. According to FTIR (Fig. 3), the α-helical concentration is more intense in the solvent-induced fibrillar structures than the temperatureinduced fibrils (Keppler et al., 2019). In addition, TEM experiments show a distinctly different morphology here than the several micrometer long fibrils forming at pH 2. Thus, it is likely that the solvent induced amyloid-like aggregates could have different functional properties than the straight BLG fibrils. 5. Conclusion The present results give evidence that ethanol-induced amyloid-like aggregates of BLG produced in similar conditions as described for temperature-induced amyloids (pH 2, 2.5% protein, low ionic strength, 350 rpm stirring) occur only at a certain solvent concentration (30% at room temperature), which is caused by the lowered hydrophobicity and interactions between the solvent and the protein. Further solvent addi tion has a detrimental effect and the aggregates can be destroyed again. Increasing temperature lowers the onset of amyloid-like aggregation considerably, probably due to its effect on the solvent polarity, protein nativity and solvent-protein interactions. The resulting aggregates are polydisperse with worm-like fibrillar structures and other spherical ag gregates and they appear already during 1–2 h incubation at room temperature and within or below 0.5 h at elevated temperature >30 � C. Temperature-induced pH 2 amyloids have already been used for microencapsulation, to stabilize interfaces in foams and emulsions as well as for film formation. Ethanol-induced amyloid-like aggregates may be suitable for microencapsulation or film formation, or generally in processes where evaporation of residual alcohol occurs. It remains to be seen how and in what time frame solvent removal, pH value changes and processing will affect their conformation and stability. However, compared to temperature-induced amyloid aggregates protein oxidation could be moderate, since only a short incubation time and a relatively low temperature is required. Therefore, it would be interesting to investigate the functional potential of ethanol-induced amyloid aggre gates (and other food grade alcohols) in the future.
4.3. Morphology of amyloid aggregates As described above, at an individual temperature-solvent concen tration, fibrillar aggregates of BLG were formed. The results of the SEC measurement (Fig. 5) give evidence that the observed aggregates consist of intact protein instead of hydrolysates, likely because acid hydrolysis within 5 h is observed at higher temperatures than used in the present manuscript (Loveday et al., 2012). For 30% ethanol at 30 � C ThT and FTIR results confirm the amyloid character of the aggregates after several h of incubation (Figs. 2 and 3). In TEM images (Fig. 4), however, structural changes to spherical aggregates were found immediately (0 h), which speaks for a rapid initial aggregation mechanism that is not the case for amyloid-like structures (see also FTIR, Fig. 3 D, E and F). The proteins form a radial structure with a size of ̴ 5–10 nm. After several hours of incubation, radial aggregates are observed together with worm-like amyloids. It can be hypothesized that ethanol leads to fast, disordered immediate aggregation (0 h incubation), which initially fa vors spherical aggregates that are likely not amyloids (i.e., no stacked beta sheets according to ThT and FTIR), while the formation of worm-like aggregates from stacked β-sheets (and probably some α-he lical elements) takes longer (1–2 h) at room temperature or 30 � C and within 0.5 h at elevated temperature. Yamaguchi and colleagues sug gested that amyloid and non-amyloid aggregation compete (Yamaguchi et al., 2006), which could explain the large amount of spherical non-amyloid aggregates here. It was found that worm-like to spherical aggregates occur near the isoelectric point of BLG (for temperature-induced amyloids), which can be attributed to a rapid association due to lack of repulsion forces (Krebs et al., 2009; Serfert et al., 2014; Keppler et al., 2019). Further, it could be shown that surfaces of short worm-like fibrils are generally more disordered and have more α-helix structures than those of long straight fibrils (VandenAkker et al., 2016). DMSO and methanol addition also results in some spherical aggre gates immediately after incubation (TEM (Fig. 4)), although far less α-helical content is observed (FTIR, Fig. 3), thus these aggregates are probably not per se helical dominated. Further studies are needed to confirm this. DMSO aggregates are slightly longer than those observed for ethanol or methanol and variances in appearance of aggregates were observed previously for different alcohols, probably caused by the different interaction kinetics with the protein (Gosal et al., 2004).
Declaration of competing interest None. Acknowledgement ASH, KS and JKK would like to thank the German Research Foun dation (DFG, Germany) for financial support of this work within the SPP 1934 priority program DiSPBiotech (project number 315456892). PA, JJK and JKK thank the research focus Kiel Life Science (KLS) for the young investigator funding. We are grateful to Monique Heuer and Jesco Reimers for competent help in the laboratory. References Akkermans, C., van der Goot, A.J., Venema, P., van der Linden, E., Boom, R.M., 2008. Formation of fibrillar whey protein aggregates. Influence of heat and shear treatment, and resulting rheology. Food Hydrocolloids 22 (7), 1315–1325. https:// doi.org/10.1016/j.foodhyd.2007.07.001. S. Akkermans, Cynthia, Venema, Paul, van der Goot, Jan, Atze, Boom, Remko M., van der Linden, Erik, 2008. Enzyme-induced formation of β-lactoglobulin fibrils by AspN endoproteinase. Food Biophys. 3 (4), 390–394. https://doi.org/10.1007/s11483008-9094-3. S.
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Functional ethanol-induced fibrils: Influence of solvents and temperature on amyloid-like aggregation of beta-lactoglobulin Jil J. Kayser a, Philipp Arnold b, Anja Steffen-Heins a, Karin Schwarz a, Julia K. Keppler a, c, * a
Institute of Human Nutrition and Food Science, Division of Food Technology, Kiel University, 24118 Kiel, Germany Anatomical Institute, Kiel University, 24118 Kiel, Germany c Laboratory of Food Process Engineering, Wageningen University, Bornse Weilanden 9, 6708WG, Wageningen, P.O. Box 17, 6700 AA, Wageningen, the Netherlands b
A R T I C L E I N F O
A B S T R A C T
Keywords: Functional fibrils Lactoglobulin Whey protein Amyloid aggregation Ethanol Solvent-induced protein denaturation
Solvent-induced fibrillar aggregates of beta-lactoglobulin occur only in a certain balance between hydrophobic forces and electrostatic interactions. We hypothesize, that different hydrophobic solvent molecules as well as rising temperatures influence this equilibrium and thus the optimum to produce amyloid aggregates. Dimethyl sulfoxide (DMSO), methanol and ethanol all resulted in polydisperse solutions with worm-like and spherical-aggregates, albeit to different degrees: the volume fraction required for aggregation was DMSO (50%) > methanol (40%) > ethanol (30%) which does not reflect their hydrophobicity. Further solvent addition decreased the fibrillar aggregation again. Increasing the temperature by 10–20 K decreased the solvent con centration needed to induce amyloid-like aggregates. A targeted production of solvent-based amyloid-like aggregates is therefore not only dependent on the hy drophobicity of the solvents, but also on their direct interaction with the protein (denaturation).
1. Introduction Among temperature-induced protein aggregates, amyloid aggregates are characterized by their stacked beta-sheet conformation, which is formed by several individual self-associating building blocks (Kroes- Nijboer et al., 2012), mainly driven by hydrophobic interactions and further stabilized by hydrogen bonds (Nicolai et al., 2011). Different types of temperature-induced functional amyloid and amyloid-like ag gregates from whey protein beta-lactoglobulin (BLG) can be distin guished by their morphology, depending on pH value, ionic strength, stirring speed, temperature, incubation time and protein concentration (Cao and Mezzenga, 2019; Keppler et al., 2019; Heyn et al., 2019; Serfert et al., 2014; Nicolai and Durand, 2013; Loveday et al., 2009; Jung and Mezzenga, 2010). Amyloid aggregates of different morphologies have increasingly become the focus of research in recent years owing to interesting functional properties that make them suitable as e.g. thick eners, gelling agents, stabilizers in emulsions and foams as well as for microencapsulation, film formation and surface coatings (Cao et al., 2019; Wei et al., 2017; Serfert et al., 2014; Humblet-Hua et al., 2013; Kroes-Nijboer et al., 2012; Li et al., 2012; Isa et al., 2011; Knowles et al.,
2010; Loveday et al., 2009; Akkermans et al., 2008a). However, incu bation during 5 h at temperatures >80 � C also results in oxidative pro tein deterioration (Keppler et al., 2019) and high production costs. Apart from temperature induced protein denaturation, other sub stances such as urea and guanidine hydrochloride as well as molecular crowding agents such as polyethylene glycol can also induce ThioflavinT reactive fibrillar aggregates (Ma et al., 2013; Akkermans et al., 2008b; Hamada and Dobson, 2002). Their assembly mechanism and structure may differ from temperature-induced amyloids observed at pH 2, which is why they are often called “amyloid-like” fibrillar assemblies. In addition, various alcohols and solvents were found to unfold proteins and stabilize the peptide structure by lowering the polarity of the solvent and binding to the protein. The alcohol-induced denaturation of the native protein is usually followed by the induction of a α-helix structure and further transformation into a predominant β-sheet conformation (Hirota-Nakaoka and Goto, 1999; Hong et al., 1999; Yoshida et al., 2012). At high protein concentrations, this mechanism can be followed by amyloid-like aggregation, which is caused by interactions between the protein molecules (Yoshida et al., 2012). Such amyloids induced by food additive ethanol could be an alternative to the
* Corresponding author.Laboratory of Food Process Engineering, Wageningen University, the Netherlands Bornse Weilanden 9, 6708WG, Wageningen. P.O. Box 17, 6700 AA, Wageningen. E-mail addresses: [email protected] (J.J. Kayser), [email protected] (P. Arnold), [email protected] (A. Steffen-Heins), info@foodtech. uni-kiel.de (K. Schwarz), [email protected] (J.K. Keppler). https://doi.org/10.1016/j.jfoodeng.2019.109764 Received 4 September 2019; Received in revised form 11 October 2019; Accepted 13 October 2019 Available online 16 October 2019 0260-8774/© 2019 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
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temperature-induced production of functional amyloids from BLG. To better understand the alcohol and solvent-induced amyloid-like aggregation, the influence of different solvents on the model protein BLG has been investigated in the past. Alcohols that have already been studied more intensively in combination with the protein are 2,2,2-tri fluoroethanol, 3,3,30 ,30 ,30 -hexalfluoro-2-propanol, methanol, ethanol and propane-2-ol with varying influencing factors such as protein con centration, pH value, temperature, alcohol concentration and incuba tion time (Hong et al., 1999; Gosal et al. 2002, 2004; Hirota-Nakaoka and Goto, 1999; Yoshida et al. 2012, 2014; Uversky et al., 1997; Mousavi et al., 2008; Maity et al., 2016). To the best of our knowledge the effect of ethanol on BLG amyloid aggregation at an acidic pH of 2.0 was investigated only at room temperature (Gosal et al., 2004; Uversky et al., 1997) while only at a pH of 7.0 a variation of temperature (25–70 � C) was included (Yoshida et al., 2014). So far, no study specif ically analyzed the effects of ethanol on BLG under similar conditions as applied for the preparation of functional temperature-induced amyloids. To investigate the functionality for the targeted production of func tional ethanol-induced BLG amyloids in the future, however, a deeper understanding of the effect of the food additive ethanol on protein ag gregation is required. For better comparability, the aggregation behavior of BLG induced by this alcohol was investigated in an envi ronment similar to the one used for temperature-induced functional amyloid preparation (i.e., 2.5% protein, pH 2, low ionic strength, 5 h incubation and gentle stirring at 350 rpm), albeit at lower temperatures between 30 � C and 50 � C. To get a better understanding of the under lying mechanisms, two further non-food grade solvents were added, one with a higher (methanol) and one with a lower polarity (DMSO) than ethanol. Similar methods typically used to study functional fibrils, such as beta-sheet stacking by Thioflavin-T test (ThT), conformation by FTIR, amyloid building blocks by size-exclusion chromatography (SEC), and morphology by TEM, are used in the experimental setup. The experiments should identify suitable combinations of incubation time, solvent concentration and temperature for the targeted production of solvent-based fibrillar aggregates that can be used in the future to keep production costs low and avoid oxidative protein damage.
2.3. Determination of amyloid aggregation 2.3.1. Thioflavin-T assay The analysis was performed according to the protocols of Loveday et al., (2012), Akkermans et al., (2008c) and Serfert et al., (2014) using a fluorescence spectrophotometer (Cary Eclipse, Varian GmbH Darmstadt, Germany). A stock solution of ThT in phosphate-NaCl buffer (10 mM phosphate and 150 mM NaCl, pH 7.0) was filtered through a 2.0 μm pore size membrane filter (Porafil, Macherey-Nagel GmbH & Co. KG, Düren, Germany) and stored in a dark bottle protected from light. The samples taken were diluted to 1 wt% with demineralized water set to pH 2.0. 48 μl of the diluted protein solution were pipetted into 4 ml of the ThT stock solution. This mixture was immediately vortexed for 20 s and measured after one minute of incubation at 440 nm excitation and 482 nm emission. For the measurement, the 1 � 1 cm quartz cu vettes with four polished sides were used. 2.3.2. Fourier-transform-infrared-spectroscopy (FT-IR) ATR-Fourier transform infrared spectroscopy (FT-IR) was performed on a Confocheck™ Tensor 2 system (Bruker Optics, Ettingen, Germany) optimized for protein analytics in solutions and a thermally controlled BioATR2 unit as described before (Keppler et al., 2019). The samples were first diluted to 1 wt% with demineralized water at pH 2.0 and the respective volume percentage solvent added. Measure ments were conducted at a temperature of 25 � C against the respective solvent mixtures without protein as background and averaged over 120 scans at a resolution of 0.7 cm 1. For evaluation, the measured spectra in the frequency range of the amide band І (1590 - 1700 cm 1) under went atmospheric correction and were then vector-normalised. The second derivative was calculated using 9 smoothing points. 2.3.3. Transmission electron microscopy (TEM) Transmission electron microscopy (TEM) was performed as described before (Arnold et al., 2014). In brief 5 μl of sample were loaded onto previously negative glow discharged continuous carbon grids (Science Service, Munich, Germany). After adsorption of the sample for 10 s, access sample was blotted off and the grid was stained with a half-saturated solution of uranyl acetate. After air drying the sample was transferred into an JEOL 1400 Plus electron microscope operating at 100 kV (JEOL, Munich, Germany). Images were acquired utilizing a F416 4kx4k digital camera (TVIPS, Munich, Germany).
2. Materials and methods 2.1. Materials Beta-Lactoglobulin (BLG) was obtained from Davisco Foods Inter national, (Inc., Eden Prairie, US) with 97% protein and 96% BLG in dry matter. Thioflavin-T (ThT >95%) was obtained from EMD Chemicals Inc.. Guanidine hydrochloride (GHCl �99.5%) was obtained from Carl Roth GmbH þ Co. KG and 1,4-dithiothreitol (DTT �99,0%) originated from Sigma Aldrich Inc. All other chemicals were of analytical grade and obtained from Sigma Aldrich Inc.
2.3.4. Size exclusion chromatography (SEC) An Agilent 1100 HPLC system equipped with a diode array detector set at 205 nm, 214 nm, 278 nm and 280 nm wavelength was used with a Superdex™ 200 increase 10/300 GL SEC column from GE healthcare (Bio-Sciences AB, Uppsala, Sweden). The samples were prepared as described in Keppler et al. (2019): 0.08 ml of the 2.5 wt% sample solution were added to 0.42 ml dithio threitol (DTT) and guanidine hydrochloride (GHCl) solution (100 mM Dithiothreitol and 8 M Guanidine dissolved in 0.15 M Tris HCl buffer (pH 8) and incubated for 60 min at room temperature under continuous shaking at 140 RPM. 0.215 ml of the 0.15 M Tris HCl buffer were added and the solution was vortexed again. The samples were then filtered through a 0.2 μl cellulose filter and 50 μl were injected in the column with 0.15 M Tris HCl buffer as eluent. The flow rate was 0.3 ml/min. The calibration kit “Gel Filtration Calibration Kit LMW” from GE Healthcare was used for calibration in similar conditions.
2.2. Preparation of BLG solutions and production of fibrils BLG was dissolved at room temperature in demineralized water. Then, a final volume of 100 ml was adjusted with demineralized solventwater so that the solution had a total protein concentration of 2.5 wt% BLG. The pH value of the solution was set to pH 2.0 with 6 M and 1 M HCl and the final solvent volume (ethanol, methanol or DMSO) was either 20%, 30%, 40% or 50%. After preparation, all samples were directly transferred to the incubation chamber based: 40 ml of the sample were placed in a Schott glass with closed screw caps (125 ml max volume) and heated for 5 h at 30 � C, 40 � C or 50 � C with constant stirring at 350 rpm using a water proof magnetic stirrer (2mag MIXdrive 6HT, 2mag AG, Munich, Germany). Every 30 min, a sample of 1 ml was taken, cooled in an ice-water bath and stored at 4 � C until further use.
2.3.5. Electron paramagnetic spectroscopy (EPR) The micro polarity of the various solvent mixtures, in concentration series of 10–70 wt% alcohol were determined by the electron para magnetic resonance spectroscopy (Bruker Elexsys II E500 spectrometer, Rheinstetten, Germany) using the spin probe Tempol0.5 mM Tempol (4 Hydroxy Tempo 97%; Sigma Aldrich) and the percentage of the solvent were added to the sample and the sample was adjusted to a pH value of 2
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2.0 with 1 M and 0.5 M HCl. The samples were measured three times. The following parameters were used for the spectra acquisition: micro wave frequency, 9.85 GHz; modulation frequency, 100 kHz; microwave power, 0.2 mW; modulation amplitude, 1.30 G; time constant, 10.24 m s; conversion time, 10.3 m s. The center field was optimized to 3511 G with a sweep width of 60 G.
(Ban et al., 2003; Xue et al., 2017). The reference sample (protein so lution without alcohol addition) as well as the one with 20% ethanol showed a constant low ThT value of ̴ 4 Int. A.U. during 5 h incubation (Fig. 2 A). However, an exchange of 30% ethanol resulted in a strong fluores cence increase already in the first 90 min (~10–70 Int. A.U.) and reached a final maximum after 5 h of 80 Int. A.U. Similar findings were evident for higher ethanol volumes, although the maximum ThT fluo rescence derived after 5 h incubation decreased with further ethanol replacement (i.e., 80 Int A.U., 60 Int A.U. and 50 Int A.U. fluorescence maximum for 30%, 40% and 50% volume replacement). A similar effect was observed for methanol (Fig. 2 B), although here the ThT-fluorescence increased significantly only with more than 30% methanol and for DMSO (Fig. 2 C), the ThT-fluorescence was only evident after more than 40% solvent replacement. The most ThT-active solvent mixtures (i.e., �30% ethanol, �40% methanol and �50% DMSO) showed a rapid increase of ThTfluorescence in all three solvents, indicating a temporal component in the solvent induced amyloid aggregation. However, methanol has the highest initial values at the beginning of incubation, whereas DMSO shows the slowest reaction.
2.3.6. Statistics Statistical analysis was conducted with the software GraphPad PRISM (version 8.2.1, GraphPad Software, San Diego, USA). A two-way ANOVA with Tukeys post hoc test was conducted at 0.05 level of significance. 3. Results 3.1. Effect of alcohol addition on solvent polarity at room temperature The nitroxide spin probe Tempol dissolved in hydro-ethanolic solu tion resulted in a typical three-line spectrum of a free tumbling nitroxide (Fig. 1). This is apparent in the same peak amplitude and peak width of all three lines as well as in the equal distance between the peaks, the socalled hyperfine splitting aN [G]. The hyperfine splitting parameter aN [G] given by the distance between the low field peak (hþ1) and the middle field peak (h0) is indicative for the micropolarity of the spin probe environment being lower in more non-polar environments. An increasing volume replacement by alcohol from 10 to 70% reduced aN, indicating a less polar environment. Table 2 lists the observed hyperfine splitting parameters for aqueous ethanolic, methanolic and DMSO so lutions at room temperature. The micropolarity can range between aN ¼ 17 [G] for an highly polar environment and aN<13 [G] for nonpolar solvents (Berliner, 1976). All solutions with 10%–30% solvent replacement were highly hy drophilic, as indicated by aN between 16.9 G and 16.8 G. Generally, the observed aN decreased with increasing volume replacement in the experimental range of 30%–50% in the order ethanol ¼ DMSO > meth anol. At 70% solvent replacement, a minimum aN of 16.27 G was observed for DMSO, while ethanol reached 16.37 G and methanol 16.48 G. This order confirms the relative polarity listed in the specifi cation (Table 1).
3.2.2. Infrared spectroscopy: increasing alcohol concentrations The influence of the solvents on the secondary structure of BLG was investigated by ATR-FT- IR before (0 h) and after incubation (5 h). The amid І region (1600-1700 cm 1) is indicative for conformational changes of the protein (Haris and Chapman, 1995; Nilsson, 2004). A typical native protein spectrum in the second derivation is shown in Fig. 3 A. Spectral band minima at 1635 cm 1 reflect intramolecular native beta-sheets, while ̴ 1655-1658 cm 1 is indicative for helical content and 1619-1620 cm 1 is often observed for intermolecular ag gregates typical for denatured and aggregated proteins and often observed in amyloid aggregates (Keppler et al., 2019; Heyn et al. 2019; Sarroukh et al., 2013; Matheus et al., 2006). In the control experimental approach with 0% volume replacement of solvent (Fig. 3A), the initially strong native β-sheet signal at 1635 cm 1 (black continuous line) did not change significantly during the 5 h incubation (black dotted line). With increasing ethanol replacement to 30%, the α-helix signal (1655-58 cm 1) increased directly after incubation at 0 h (Fig. 3 D), then decreased at the expense of a strong signal at 1619 cm 1 in the 5 h incubated samples, indicating a fast initial helix formation followed by intermolecular β-sheets typical for amyloids or aggregated protein which were not present at the beginning of the incubation (0 h). This signal was also present at 40% and 50% alcohol replacement although its intensity decreased from 30%
3.2. Influence of alcohol and temperature on amyloid aggregation 3.2.1. Thioflavin-T fluorescence: increasing alcohol concentration ThT fluorescence is frequently used for analysis of amyloid aggre gates because the ThT molecule binds tightly to assembled beta-sheets
Fig. 1. Exemplary electron paramagnetic resonance spectra of spin probe tempol with 10% or 70% hydro-ethanolic solution at room temperature. The line indicates the distance between the low and the middle field peaks. 3
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Table 1 Physico-chemical properties of the three solvents. Molar mass [g/mol] Density at 20 � C [g/cm3] 30% volume exchange [g/g water] 30% volume exchange [M/M water] Miscibility with water Relative polarity Boiling point [� C] Molecular structure
Ethanol
Methanol
DMSO
46.07 0.78–0.81 ~0.33–0.34 ~0.13 Fully miscible 0.654 78
32.04 0.79 ~0.35 ~0.2 Fully miscible 0.762 65
78.13 1.1 ~0.47 ~0.1 100 g/l 0.444 189
much higher length of about 100–150 nm.
Table 2 Hyperfine splitting parameters aN [G] for solvent-water mixtures at room tem perature. Values indicated by different superscript letters (a to c) are signifi cantly different in a row, p 0.05. All measurements were conducted in triplicate and listed as mean and standard deviation. Solvent (%)
Ethanol
Methanol
3.2.4. Size exclusion chromatography: increasing alcohol concentration The results confirm the presence of BLG monomers with a size of ̴ 19.1 kDa ( ̴ 51 min elution time according to calibration, monomeric BLG has a theoretical size of 18.4 kDa) and some aggregates ( ̴40 min elution time). These aggregates could be dimers. No clear evidence of protein hydrolysis during incubation can be observed (Fig. 5 A, B and C): Protein hydrolysates with a size smaller than monomeric BLG would have led to signals with higher elution times (>51 min).
DMSO
Micropolarity aN [G] 10 20 30 40 50 60 70
16.93 16.89 16.76 16.70 16.54 16.46 16.37
�0.00 a �0.03 a �0.00 b �0.06 a �0.03 b �0.06 b �0.03 b
16.91 16.86 16.84 16.76 16.64 16.60 16.48
�0.03 �0.03 �0.03 �0.00 �0.00 �0.03 �0.03
a a a a a a a
16.91 16.88 16.82 16.70 16.56 16.43 16.27
�0.03 a �0.00 a �0.00 a �0.06 a �0.03 b �0.03 b �0.03 c
3.3. Influence of temperature and alcohol on amyloid aggregation 3.3.1. Thioflavin-T fluorescence: increasing temperature In a second experimental approach, the influence of temperature on the aggregation behavior of BLG in solvent solutions were tested. For ethanol (Fig. 6 A), increasing the incubation temperature from 30 � C, to 40 � C, or 50 � C increased the aggregation speed significantly. A ThT fluorescence of 70 Int A.U. is reached in 1.5 h while it takes 1 h at 40 � C and 0.5 h at 50 � C. However, it seems that further incubation has a detrimental effect especially at 50 � C and the fluorescence decreases again to 60 Int. A.U. For methanol (Fig. 6 B) a similar effect can be observed although to a much stronger extent. Here, almost no ThT reactivity is evident at 30 � C, while it increases strongly at 40 � C and 50 � C. Again, further incubation to 5 h reduces the fluorescence slightly. For DMSO (Fig. 6 C) ThT fluorescence is also positively affected by increasing temperature, although here �50 � C are needed to obtain any significant signal.
to 40%–50% ethanol (Fig. 3 F). The native β-sheet signal decreased continuously after surpassing 20% ethanol (Fig. 3 E). For methanol (Fig. 3 B, D, E and F) a similar effect as for ethanol can be observed, although here, a first shift to the 1619 cm 1 waveband is only evident at 40% alcohol replacement and also only after 5 h incu bation. Likewise, the intensity of the intramolecular β-sheets decreased continuously with increasing methanol addition, the α-helical signal increased only initially (0 h) and to a lower extent than observed for ethanol, this effect was no longer evident after 5 h incubation. With DMSO (Fig. 3 C, D, E and F), a large volume exchange of 50% is necessary to induce intermolecular beta-sheets at 30 � C after 5 h incu bation. Below that, DMSO seems to have no strong effect on the helix or native β-sheet conformation, since 0 h and 5 h signals were not signifi cantly different except for a native β-sheet increase at 30% (Fig. 3 E). Only at a volume exchange of 50% DMSO noteworthy conforma tional changes occur such as the strong signal for intermolecular betasheets at 1619 cm 1.
3.3.2. Infrared spectroscopy: increasing temperature ATR-FTIR spectra confirm the findings of ThT. Between 30 � C and 50 � C a strong signal for the intermolecular beta-sheet aggregation at 1619 cm 1 is evident for ethanol (Fig. 7 A). For methanol this signal only occurs for temperatures similar or higher than 40 � C and for DMSO a first effect can be observed only at 50 � C.
3.2.3. Transmission electron microscopy: increasing alcohol concentration To confirm the presence of fibrillar protein aggregates, representa tive TEM images of the samples with 30% volume exchange by solvents were taken (Fig. 4A). Directly after solvent exchange (0 h) ethanol and DMSO samples show spherical aggregates of approximately 20–25 nm size. After 5 h incubation at 30 � C the TEM images of the sample give evidence that the morphology of the formed amyloid aggregates is short and worm-like with a length of ̴ 20–60 nm as well as partly radial. Similar aggregates can be observed in methanol, albeit significantly less pronounced. In DMSO only few aggregates are present, however, already at 0 h some spherical aggregates can be observed. In addition, also for a volume exchange with 40% methanol and 50% DMSO TEM images were taken (Fig. 4 B). After 5 h incubation at 30 � C the samples show also amyloid aggregates. A high proportion of amyloid aggregates can be observed in the sample with methanol, also with a short worm-like structure and a length of approximately 15–40 nm. In the solvent DMSO only a few amyloid aggregates are present but with a
4. Discussion 4.1. Influence of increasing volume exchange with solvents on amyloid aggregation 4.1.1. Maximum amyloid aggregation The results give evidence that all tested solvents influenced the amyloid-like self-association of BLG at pH 2 as determined by ThT (Fig. 2), FTIR (Fig. 3) and TEM (Fig. 4), albeit to a different extent: The alcohol or solvent volume fraction needed for inducing amyloidlike aggregation was in the order DMSO (50%) > methanol (40%) > ethanol (30%) (Fig. 4B). The individual concentration required to induce aggregation can be traced back to several factors: If the polarity of the two alcohols methanol and ethanol is compared 4
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Fig. 2. Thioflavin-T fluorescence during 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol, B) Methanol und C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean and standard deviation of three samples.
(Table 2), it can be observed that a volume exchange of 30% ethanol results in the same polarity as a volume exchange of 40% methanol. A volume exchange of 50% ethanol and 50% DMSO, which have approximately the same ThT values after the end of the incubation period (Fig. 2), also shows a very similar polarity. A lower polarity of the solvent compared to water leads to a weakening of the hydrophobic interactions as well as to a strengthening of the electrostatic interactions (Hong et al., 1999; Hirota-Nakaoka and Goto, 1999; Nikolaidis et al., 2017). The altered equilibrium can result in a conformational change into the α-helix structure (Hirota-Nakaoka and Goto, 1999; Hong et al., 1999; Yoshida et al., 2012), which is also evident in FTIR (Fig. 3) where helix formation increased immediately after incubation at elevated solvent exchange for ethanol and methanol. Similar to the present findings, for ethanol it is known that a conformational transition to the α-helix structure takes place from a solvent concentration of 20% on wards (Nikolaidis et al., 2017). This helix transition is usually followed by a further transition to amyloid-like β-sheets at higher solvent concentrations (Yoshida et al., 2012). Interestingly, the ThT graphs (Fig. 2) show that in contrast to the rapid initial helix formation, the beta-sheet transition for 30% ethanol or 40% methanol takes approximately 1–2 h. This time difference between helix formation and beta-sheet transformation is also confirmed by the FTIR results, although only samples after 0 h and 5 h incubation were measured here (Fig. 3). Thus, the intermolecular β-sheet formation
initially follows slower kinetics than α-helix formation, although this effect is less pronounced with higher solvent addition, at least in the ThT test experiments. The stronger effect of ethanol than methanol on polarity can be traced back to the hydroxyl group having a negative effect on the described alcohol-induced effect. Thus the alcohol concentration required to induce the conformational transition decreases with increasing size of the alcohol molecule (Hirota-Nakaoka and Goto, 1999) (i.e., MW 46.06 for ethanol and 32.04 for methanol). In addition, some alcohols have a propensity to form micelles or clusters in aqueous solutions, which was found to further increase the observed effect on proteins (Hirota-Nakaoka and Goto, 1999). The organosulfur solvent DMSO has not been studied with respect to amyloid-like aggregation behavior of BLG and it acts differently than the alcohols. The sample with a volume exchange of 40% DMSO has the same polarity as the sample with 40% ethanol, but still a considerably lower effect on helix formation or amyloid-like aggregation was observed by ThT (Fig. 2 C) and FTIR (Fig. 3 C). Looking at the FT-IR spectra of the test series with DMSO (Fig. 3 C), it is evident that con trary to both alcohols, DMSO has no strong denaturing effect on BLG at low temperatures of 30 � C and low volume concentrations of up to 40%. Only from a volume exchange of 50% by DMSO, conformational tran sition can be observed. A preferential hydration of BLG was evident in the presence of low DMSO concentrations <50% volume exchange in 5
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Fig. 3. FTIR second derivation spectra before and after 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol B) Methanol C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean of three individual measurements. A summary of D) the α-helix development, E) the native β-sheets and F) the intermolecular “amyloid” β -sheet development after 0 and 5 h incubation for each solvent and solvent concentration is given. The data are readouts of the above listed mean FTIR spectra and are thus only sketches without standard deviation to emphasize the temporal development.
6
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Fig. 4. TEM images of the respective samples directly after preparation (0 h) and after 5 h incubation at 30 � C A) with solvent solutions of 30% for ethanol, methanol and DMSO and B) solvent solutions with 40% for methanol and 50% for DMSO to confirm the presence of amyloid-like aggregates. Small inserts show a range of 200 nm.
B
400
0h 2h 5h
Intensity [A.U.]
Intensity [A.U.]
600
600
200
0
400
50
time [min]
60
0h 2h 5h
200
0 40
C 600
Intensity [A.U.]
A
40
50
time [min]
60
400
0h 2h 5h
200
0
40
50
time [min]
60
Fig. 5. Size exclusion chromatograms of dissociated BLG aggregates which were incubated in 30% solvent-water mixtures for 0 h, 2 h or 5 h at 30 C. A) ethanol, B) methanol and C) DMSO.
7
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Fig. 6. Thioflavin-T fluorescence during 5 h incubation of beta-Lactoglobulin at 30 � C (2.5% Protein, pH 2.0 & 350 RPM). (A) Ethanol, B) Methanol und C) Dimethyl Sulfoxide. The alcohol concentration was either 0%, 20%, 30%, 40% or 50% as indicated above each figure section. All measurements are listed as mean and standard deviation.
Fig. 7. FTIR second derivation spectra after 5 h incubation of beta-lactoglobulin with a solvent concentration of 30% (2.5% protein & 350 RPM). (A) ethanol (B) methanol (C) Dimethyl sulfoxide. The temperature was either 30 � C, 40 � C or 50 � C. All measurements are listed as mean of three individual measurements.
water, which resulted in preservation of the proteins native structure (Arakawa et al., 2007). In addition, because of the larger size (approx. 80 Da) DMSO is less able than ethanol to penetrate the native molecule in order to compete with non-polar small-range bonds. It is also evident from FTIR (Fig. 3) that intermolecular β-sheet formation of BLG is less pronounced in the presence of DMSO, which also confirms that there is less direct interaction between the hydrophobic groups of DMSO with those of the protein at low DMSO concentrations. However, >50% DMSO replacement finally has a denaturing effect on the protein. Only after this opening of the tertiary structure DMSO can interact and sta bilize the secondary conformation of BLG and lead to the observed larger amyloid-like aggregates formed in the presence of 50% solvent exchange with DMSO compared to ethanol and methanol (Arakawa et al., 2007; Inoue and Timasheff, 1972).
hydrophobic interactions initiate protein denaturation at lower tem perature. On the other hand, amyloid-like aggregates initially associate by hydrophobic forces and are then stabilized by hydrogen bonds. Therefore, it is likely that any further aggregation above a certain hy drophobicity (solvent concentration) is reduced, while the increasing hydrogen bond stability favors the stability of the formed fibrils. 4.2. Effect of temperature on solvent induced amyloid aggregation To alter the delicate balance between hydrophobic forces and hydrogen bonds further, the following experiments were conducted at a fixed solvent exchange of 30% but with increasing temperature. As ex pected, higher temperatures can lower the threshold for the solvent induced amyloid-like aggregation up to a certain point, where further temperature increase than again also has a detrimental effect (Figs. 6 and 7). It is well known that increasing temperature decreases the dielectric constant of the solution (Suryanarayana and Govindaswamy, 1961). In the previous section it could be shown that at a polarity induced by a volume exchange of 30% by ethanol and a temperature of 30 � C, con ditions are present which seem to promote the formation of amyloid-like aggregates. If the polarity of the medium in 30% methanol were still too high to induce fibrillar aggregates (Table 2), increasing the temperature from 30 � C to 40 � C could reduce the polarity of the medium and thus restore the balance between hydrophobic and electrostatic interactions favored by amyloid-like aggregates in a simplified model. A further in crease in temperature further alters the balance between hydrophobic and electrostatic interactions, so that the formation of fibrillar aggre gates is then again less favorable. Hence, this could be one possible
4.1.2. Decreasing amyloid aggregation with increasing solvents There seems to be an optimum solvent concentration to initiate amyloid-like aggregation here. As is evident in ThT (Fig. 2) and FTIR (Fig. 3), solvent concentrations above a certain optimum result in a decline of the amyloid-like aggregation effects (i.e., �40% for ethanol, �50% for methanol and not measured for DMSO), while the α-helix conformation increases linearly (Fig. 3D) only in ethanol. Yamaguchi and coworkers showed that the TFE-induced amyloid aggregation of a microglobulin peptide followed a similar bell shape as observed in the present manuscript for BLG, and thus reached an opti mum after which an alpha-helical-conformation dominated again. They argued that this was due to a sensitive balance between decreasing hy drophobic and increasing polar interactions with increasing solvent concentration (Yamaguchi et al., 2006). On the one hand, decreasing 8
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explanation of the observed effects. Elevated temperatures could also enhance the solvent-protein in teractions due to the altered solvent hydrophobicity as well as due to increased molecular mobility of water and solvent. In addition, the molecular flexibility of BLG improves the accessibility of its non-polar amino acids to the solvent molecules. This would result in an acceler ated exchange of surface bound water molecules by solvent molecules via short range hydrophobic interactions (Chong et al., 2015) and consequently exert an effect on the protein β-sheet formation (Yoshida et al., 2014). This would especially explain the enhanced aggregation observed with DMSO at 50 � C, where a strong conformational change is evident in FTIR with 30% solvent replacement (Fig. 7), while this is not observed at lower temperatures. There are also reports about a lowered denaturation temperature of proteins in the presence of solvents (Glu giarelli et al. 2012), while normally, the whey protein would be dena tured at temperatures of ̴ 70 � C (Singh et al., 2014; Keppler et al., 2014). According to the present findings, the following temperature in crease will lead to a maximum amyloid-like aggregation at 30% solvent exchange (at the given protein concentration and pH value): For methanol, the temperature would have to be increased by 20 K (from 20 to 40 � C). For DMSO a temperature increase of 40 K is probably required (from 20 to 60 � C). For ethanol it can be assumed that a temperature increase >10 K reduces the required solvent concentration to <30%.
4.4. Comparison with temperature-induced amyloid aggregates of BLG The ThT values of approximately 80 Int A.U. observed here (Fig. 2) are in the same range as described for temperature-induced amyloid aggregates, however, due to the polydispersity of the solvent induced aggregate (worm-like and spherical), the fibrillar material could not be clearly separated from non-amyloid material by centrifugal ultrafiltra tion (results not shown), so that no conversion rate can be calculated here. According to FTIR (Fig. 3), the α-helical concentration is more intense in the solvent-induced fibrillar structures than the temperatureinduced fibrils (Keppler et al., 2019). In addition, TEM experiments show a distinctly different morphology here than the several micrometer long fibrils forming at pH 2. Thus, it is likely that the solvent induced amyloid-like aggregates could have different functional properties than the straight BLG fibrils. 5. Conclusion The present results give evidence that ethanol-induced amyloid-like aggregates of BLG produced in similar conditions as described for temperature-induced amyloids (pH 2, 2.5% protein, low ionic strength, 350 rpm stirring) occur only at a certain solvent concentration (30% at room temperature), which is caused by the lowered hydrophobicity and interactions between the solvent and the protein. Further solvent addi tion has a detrimental effect and the aggregates can be destroyed again. Increasing temperature lowers the onset of amyloid-like aggregation considerably, probably due to its effect on the solvent polarity, protein nativity and solvent-protein interactions. The resulting aggregates are polydisperse with worm-like fibrillar structures and other spherical ag gregates and they appear already during 1–2 h incubation at room temperature and within or below 0.5 h at elevated temperature >30 � C. Temperature-induced pH 2 amyloids have already been used for microencapsulation, to stabilize interfaces in foams and emulsions as well as for film formation. Ethanol-induced amyloid-like aggregates may be suitable for microencapsulation or film formation, or generally in processes where evaporation of residual alcohol occurs. It remains to be seen how and in what time frame solvent removal, pH value changes and processing will affect their conformation and stability. However, compared to temperature-induced amyloid aggregates protein oxidation could be moderate, since only a short incubation time and a relatively low temperature is required. Therefore, it would be interesting to investigate the functional potential of ethanol-induced amyloid aggre gates (and other food grade alcohols) in the future.
4.3. Morphology of amyloid aggregates As described above, at an individual temperature-solvent concen tration, fibrillar aggregates of BLG were formed. The results of the SEC measurement (Fig. 5) give evidence that the observed aggregates consist of intact protein instead of hydrolysates, likely because acid hydrolysis within 5 h is observed at higher temperatures than used in the present manuscript (Loveday et al., 2012). For 30% ethanol at 30 � C ThT and FTIR results confirm the amyloid character of the aggregates after several h of incubation (Figs. 2 and 3). In TEM images (Fig. 4), however, structural changes to spherical aggregates were found immediately (0 h), which speaks for a rapid initial aggregation mechanism that is not the case for amyloid-like structures (see also FTIR, Fig. 3 D, E and F). The proteins form a radial structure with a size of ̴ 5–10 nm. After several hours of incubation, radial aggregates are observed together with worm-like amyloids. It can be hypothesized that ethanol leads to fast, disordered immediate aggregation (0 h incubation), which initially fa vors spherical aggregates that are likely not amyloids (i.e., no stacked beta sheets according to ThT and FTIR), while the formation of worm-like aggregates from stacked β-sheets (and probably some α-he lical elements) takes longer (1–2 h) at room temperature or 30 � C and within 0.5 h at elevated temperature. Yamaguchi and colleagues sug gested that amyloid and non-amyloid aggregation compete (Yamaguchi et al., 2006), which could explain the large amount of spherical non-amyloid aggregates here. It was found that worm-like to spherical aggregates occur near the isoelectric point of BLG (for temperature-induced amyloids), which can be attributed to a rapid association due to lack of repulsion forces (Krebs et al., 2009; Serfert et al., 2014; Keppler et al., 2019). Further, it could be shown that surfaces of short worm-like fibrils are generally more disordered and have more α-helix structures than those of long straight fibrils (VandenAkker et al., 2016). DMSO and methanol addition also results in some spherical aggre gates immediately after incubation (TEM (Fig. 4)), although far less α-helical content is observed (FTIR, Fig. 3), thus these aggregates are probably not per se helical dominated. Further studies are needed to confirm this. DMSO aggregates are slightly longer than those observed for ethanol or methanol and variances in appearance of aggregates were observed previously for different alcohols, probably caused by the different interaction kinetics with the protein (Gosal et al., 2004).
Declaration of competing interest None. Acknowledgement ASH, KS and JKK would like to thank the German Research Foun dation (DFG, Germany) for financial support of this work within the SPP 1934 priority program DiSPBiotech (project number 315456892). PA, JJK and JKK thank the research focus Kiel Life Science (KLS) for the young investigator funding. We are grateful to Monique Heuer and Jesco Reimers for competent help in the laboratory. References Akkermans, C., van der Goot, A.J., Venema, P., van der Linden, E., Boom, R.M., 2008. Formation of fibrillar whey protein aggregates. Influence of heat and shear treatment, and resulting rheology. Food Hydrocolloids 22 (7), 1315–1325. https:// doi.org/10.1016/j.foodhyd.2007.07.001. S. Akkermans, Cynthia, Venema, Paul, van der Goot, Jan, Atze, Boom, Remko M., van der Linden, Erik, 2008. Enzyme-induced formation of β-lactoglobulin fibrils by AspN endoproteinase. Food Biophys. 3 (4), 390–394. https://doi.org/10.1007/s11483008-9094-3. S.
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