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Amyloid formation using 1-butyl-3-methyl-imidazolium-based ionic liquids
Analytical Biochemistry 419 (2011) 354–356
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Amyloid formation using 1-butyl-3-methyl-imidazolium-based ionic liquids Song Yi Bae a, Seulgi Kim a, Bun Yeoul Lee a, Kyeong Kyu Kim b, T. Doohun Kim a,⇑ a b
Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, South Korea Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea
a r t i c l e
i n f o
Article history: Received 5 July 2011 Received in revised form 1 August 2011 Accepted 2 August 2011 Available online 10 August 2011
a b s t r a c t Amyloid fibrils are highly organized protein filaments that can be used as novel biomaterials. In this study, we show that proteins could be selectively induced to form amyloid fibrils at room temperature by the introduction of imidazolium salts, which could trigger the self-assembly process with their hydrophobic and ionic properties. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Amyloid fibrils Ionic liquid Biomaterials
Filamentous amyloid structures are normally associated with several neurodegenerative diseases such as Alzheimer’s and Parkinson’s [1,2]. These amyloid fibrils are formed by the self-assembly of naturally occurring disease-related proteins that finally aggregate into insoluble structures. To date, approximately 30 different proteins are known to self-assemble into amyloid fibrils in vivo, although there are few similarities among them regarding primary sequence, protein function, and/or tertiary structure. Amyloid fibrils are composed of antiparallel b-sheets whose orientations are perpendicular to the longitudinal fibril axis [3]. The direct growth of amyloid fibrils in vitro has provided opportunities for understanding the process of amyloid fibril formation as well as for searching for therapeutic compounds. It is now well known that the ability to form amyloid fibrils is an intrinsic property of any polypeptide, and the amyloid fold is naturally found in many structures such as adhesions, hyphae, and secretory granules [1,4,5]. Furthermore, these fibrils have attracted growing interest as biomaterials due to their remarkable strength, elasticity, and stability [6,7]. Therefore, many groups have been devoted to fabricating amyloid fibrils in vitro, and amyloid fibrils are usually obtained under specific conditions such as high temperature, low pH, high ionic strength, and oxidative stresses that favor partial unfolding of native proteins. Room temperature ionic liquids (RTILs)1 have been recently identified as useful solvents and additives for organic synthesis, elec⇑ Corresponding author. Fax: +82 31 219 2394. E-mail address: [email protected] (T.D. Kim). Abbreviations used: RTIL, room temperature ionic liquid; [BMIM], 1-butyl-3methyl-imidazolium; ThT, thioflavin T; TEM, transmission electron microscopy; a-lac, a-lactalbumin; FSC, forward scattering; SSC, side scattering; HEWL, in hen egg white lysozyme. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.08.007
troanalytical analysis, and chromatography [8,9]. In previous studies, several groups, including our group, showed that these organic salts can effectively promote or alter the self-assembly process of proteins [10–12]. In this report, we show that 1-butyl-3-methyl-imidazolium ([BMIM])-based ionic liquids (i.e., [BMIM] with different negative ions) can effectively trigger amyloid fibril formation. In a previous report, anion variation of cosolvents was shown to have a more dramatic effect on protein refolding than variation of the cations [13]. The thioflavin T (ThT) assay, flow cytometry, fluorescence microscopy, transmission electron microscopy (TEM), and other biochemical methods have been used to explore the effects of the addition of [BMIM]-based ionic liquids on amyloid fibril formation. In this study, apo a-lactalbumin (a-lac), a small Ca2+-binding protein, was used as a model protein [14] to monitor the aggregation in the presence of 5% [BMIM]-based RTILs. The effect of 5% (v/v) RTILs on the kinetics of amyloid fibril formation was evaluated by ThT assay [11,15]. Briefly, protein samples (1 mg/ml final concentration) with RTILs were incubated in 20 mM glycine buffer (pH 2.0) with constant shaking at room temperature [15]. To monitor amyloid fibril formation, 10-ll aliquots were removed at various time points during aggregation and mixed with 190 ll of ThT (10 lm final concentration in the same buffer). ThT fluorescence was measured at kex = 450 nm and kem = 490 nm after equilibrium using a spectrofluorometer (FP-6200, Jasco, Tokyo, Japan). As shown in Fig. 1A, high ThT readings indicated that amyloid fibrils were effectively formed. Although protein only and protein with [BMIM][(CF3SO2)2N] were ineffective, [BMIM][BF4] and [BMIM][PF6] were highly successful in amyloid formation with high ThT values. Specifically, [BMIM][BF4] produced highly ThT-positive amyloid fibrils compared with [BMIM][PF6]. Therefore, amyloid formation of a-lac was substantially affected by the chemical nature of
Notes & Tips / Anal. Biochem. 419 (2011) 354–356
355
Fig.1. Biochemical characterization of the amyloid formation of a-lac with [BMIM]-based ionic liquids. (A) ThT fluorescence as a function of time of apo a-lac alone (d) and in the presence of [BMIM][BF4] (D), [BMIM][(CF3SO2)2N] (j), or [BMIM][PF6] (s). (B) Fluorescence image of ThT-stained a-lac amyloid fibrils with [BMIM][BF4]. (C) Flow cytometric analysis of a-lac amyloid fibrils. Typical scattering pattern (FSC vs. SSC) is shown for protein only and for protein with [BMIM][(CF3SO2)2N], [BMIM][BF4], or [BMIM][PF6].
negative ions. In the absence of these ionic liquids, no detectable changes of fluorescence were observed at room temperature (Fig. 1A). The order of amyloid fibril-forming ability for a-lac was shown as BF4 > PF6 > (CF3SO2)2N , CH3SO4 . Amyloid fibrils induced by [BMIM][BF4] were clearly observable by a fluorescence microscope equipped with a 100-W mercury lamp using a BP filter of 470–500 nm (Fig. 1B). A FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 488-nm argon laser for forward scattering (FSC) and side scattering (SSC) was used for flow cytomet-
ric analysis. Sensitivity and gain (sensitivity = 400, gain = 20 [10 for FSC and SSC]) were optimized for the detection of large aggregates or amyloid fibrils. A linear relationship of FSC to SSC, a characteristic of amyloid fibrils [16], was clearly observed with amyloid fibrils prepared with [BMIM][BF4]. However, aggregates formed with [BMIM][(CF3SO2)2N] showed a pattern of high SSC (P200) with low FSC (6300), indicating the existence of large amorphous aggregates with no specific conformation. Interestingly, amyloid fibrils prepared with [BMIM][PF6] showed a unique distribution pattern
Fig.2. (A–C) Chemical structures of [BMIM]-based ionic liquids and TEM images of a-lac amyloid fibrils induced by [BMIM][(CF3SO2)2N] (A), [BMIM][BF4] (B), and [BMIM][PF6] (C). (D) Chemical structure of [BMIM][CH3SO4] (left) and pH-dependent TEM images of a-lac with [BMIM][CH3SO4] at 20 mM glycine (pH 2.0) (middle) and at 10 mM Tris–HCl (pH 8.0) (right). Scale bars correspond to 100 nm.
356
Notes & Tips / Anal. Biochem. 419 (2011) 354–356
with a broad range of SSC values that was different from [BMIM][BF4]. There was only a slight amount (<2.0%) of small aggregates in the absence of RTILs. Next, we compared the morphological features of [BMIM]based ionic liquid-induced amyloid fibrils using TEM (Fig. 2). Consistent with the ThT assay and flow cytometry analysis, many small aggregates with no fibrils were observed for [BMIM][(CF3SO2)2N] (Fig. 2A). For [BMIM][BF4] , amyloid fibrils of hundreds of nanometers length, but no small aggregates, were observed. Typical long straight fibrils with heights of 8–10 nm (7–12 nm in diameter) are usually seen in the presence of [BMIM][BF4] (Fig. 2B). Such fibrils have not been observed to form in the absence of ionic liquids. Interestingly, the amyloid fibrils formed with [BMIM][PF6] displayed short rod-like forms (Fig. 2C). These amyloid fibrils were very stable and resistant to heat treatment. Even incubation at 95 °C for 1 h did not produce any apparent disaggregation as determined by the ThT assay (data not shown). Furthermore, filial structures seeded with 2% parental forms displayed a very similar morphology to their initial shapes (data not shown). In the case of [BMIM][CH3SO4], only small globular aggregates were formed at pH 2.0 (20 mM glycine), whereas amyloid fibrils were clearly observed at pH 8.0 (10 mM Tris–HCl). This pH-responsive behavior was confirmed by TEM studies (Fig. 2D) and flow cytometry analysis (data not shown). Therefore, an ionic liquid, at least in the case of [BMIM][CH3SO4], could induce formation of amyloid fibrils or amorphous aggregates depending on the environmental pH. To ensure that the amyloid fibril-inducing effects of [BMIM]based ionic liquids were not dependent on the structure of a-lac, several other proteins were investigated with these ionic liquids. As shown in Fig. S1 in the supplementary material, these ionic liquids, especially [BMIM][BF4], promoted the formation of amyloid fibrils in hen egg white lysozyme (HEWL), calcium-saturated a-lac, insulin, and trypsin. In addition, it has been reported that [BMIM][(CF3SO2)2N] induced the appearance of amyloid fibrils in a-synuclein [11,15]. In general, optimizing fibril formation for different proteins will require trying different anions of [BMIM]based ionic liquids and different pH conditions. Consequently, it is clear that [BMIM]-based ionic liquids are highly effective for the preparation of amyloid fibrils. In summary, this report has presented a novel method to prepare different protein aggregates using [BMIM]-based ionic liquids as cosolvents. Furthermore, the morphology or properties of these aggregates can be modulated depending on the chemical structures of the ionic liquid or pH of the environment [17,18]. In addition, the effects of [BMIM]-based ionic liquids could be further regulated by chemical modifications or cross-linking reactions. The properties of ionic liquids presented in this study have potential uses in a broad spectrum of applications involving protein nanostructures or biopolymers.
Acknowledgments This work was supported by a Korean Research Foundation Grant funded by the Korean Government (KRF-2009-0089832) and by a research grant from Ajou University to T.D.K. K.K.K. was supported by the Korea Healthcare Technology R&D Project (A092006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2011.08.007. References [1] J. Greenwald, R. Riek, Biology of amyloid: structure, function, and regulation, Structure 18 (2010) 1244–1260. [2] V.N. Uversky, D. Elizer, Biophysics of Parkinson’s disease: structure and aggregation of a-synuclein, Curr. Protein Pept. Sci. 10 (2009) 483–499. [3] R. Nelson, D. Eisenberg, Structural models of amyloid-like fibrils, Adv. Protein Chem. 73 (2006) 235–282. [4] F. Shewmaker, R.P. McGlinchey, R.B. Wickner, Structural insights into functional and pathological amyloid, J. Biol. Chem. 286 (2011) 16533–16540. [5] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333–366. [6] I. Cherny, E. Gazit, Amyloids: not only pathological agents but also ordered nanomaterials, Angew. Chem., Int. Ed. Engl. 47 (2008) 4062–4069. [7] J.W. Kelly, W.E. Balch, Amyloid as a natural product, J. Cell Biol. 161 (2003) 461–462. [8] M. Petkovic, K.R. Seddon, L.P. Rebelo, C. Silva Pereira, Ionic liquids: a pathway to environmental acceptability, Chem. Soc. Rev. 40 (2011) 1383–1403. [9] C. Hardacre, J.D. Holbrey, M. Nieuwenhuyzen, T.G. Youngs, Structure and solvation in ionic liquids, Acc. Chem. Res. 40 (2007) 1146–1155. [10] N. Byrne, C.A. Angell, Formation and dissolution of hen egg white lysozyme amyloid fibrils in protic ionic liquids, Chem. Commun. (2009) 1046–1048. [11] H. Hwang, H. Choi, H.K. Kim, H. Jo, T.D. Kim, Ionic liquids promote amyloid formation from a-synuclein, Anal. Biochem. 386 (2009) 293–295. [12] H.R. Kalhor, M. Kamizi, J. Akbari, A. Heydari, Inhibition of amyloid formation by ionic liquids: ionic liquids affecting intermediate oligomers, Biomacromolecules 10 (2009) 2468–2475. [13] R. Buchfink, A. Tischer, G. Patil, R. Rudolph, C. Lange, Ionic liquids as refolding additives: variation of the anion, J. Biotechnol. 150 (2010) 64–72. [14] D. Kurouski, W. Lauro, I.K. Lednev, Amyloid fibrils are ‘‘alive’’: spontaneous refolding from one polymorph to another, Chem. Commun. 46 (2010) 4249– 4251. [15] S.Y. Bae, S. Kim, H. Hwang, H.K. Kim, H.C. Yoon, J.H. Kim, S. Lee, T.D. Kim, Amyloid formation and disaggregation of a-synuclein and its tandem repeat (a-TR), Biochem. Biophys. Res. Commun. 400 (2010) 531–536. [16] J. Wall, A. Solomon, Flow cytometric characterization of amyloid fibrils, Methods Enzymol. 309 (1999) 460–466. [17] R. Kodali, R. Wetzel, Polymorphism in the intermediates and products of amyloid assembly, Curr. Opin. Struct. Biol. 17 (2007) 48–57. [18] J.S. Pedersen, C.B. Andersen, D.E. Otzen, Amyloid structure—one but not the same: the many levels of fibrillar polymorphism, FEBS J. 277 (2010) 4591– 4601.
Contents lists available at SciVerse ScienceDirect
Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio
Notes & Tips
Amyloid formation using 1-butyl-3-methyl-imidazolium-based ionic liquids Song Yi Bae a, Seulgi Kim a, Bun Yeoul Lee a, Kyeong Kyu Kim b, T. Doohun Kim a,⇑ a b
Department of Molecular Science and Technology, Graduate School of Interdisciplinary Programs, Ajou University, Suwon 443-749, South Korea Department of Molecular Cell Biology, Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, Suwon 440-746, South Korea
a r t i c l e
i n f o
Article history: Received 5 July 2011 Received in revised form 1 August 2011 Accepted 2 August 2011 Available online 10 August 2011
a b s t r a c t Amyloid fibrils are highly organized protein filaments that can be used as novel biomaterials. In this study, we show that proteins could be selectively induced to form amyloid fibrils at room temperature by the introduction of imidazolium salts, which could trigger the self-assembly process with their hydrophobic and ionic properties. Ó 2011 Elsevier Inc. All rights reserved.
Keywords: Amyloid fibrils Ionic liquid Biomaterials
Filamentous amyloid structures are normally associated with several neurodegenerative diseases such as Alzheimer’s and Parkinson’s [1,2]. These amyloid fibrils are formed by the self-assembly of naturally occurring disease-related proteins that finally aggregate into insoluble structures. To date, approximately 30 different proteins are known to self-assemble into amyloid fibrils in vivo, although there are few similarities among them regarding primary sequence, protein function, and/or tertiary structure. Amyloid fibrils are composed of antiparallel b-sheets whose orientations are perpendicular to the longitudinal fibril axis [3]. The direct growth of amyloid fibrils in vitro has provided opportunities for understanding the process of amyloid fibril formation as well as for searching for therapeutic compounds. It is now well known that the ability to form amyloid fibrils is an intrinsic property of any polypeptide, and the amyloid fold is naturally found in many structures such as adhesions, hyphae, and secretory granules [1,4,5]. Furthermore, these fibrils have attracted growing interest as biomaterials due to their remarkable strength, elasticity, and stability [6,7]. Therefore, many groups have been devoted to fabricating amyloid fibrils in vitro, and amyloid fibrils are usually obtained under specific conditions such as high temperature, low pH, high ionic strength, and oxidative stresses that favor partial unfolding of native proteins. Room temperature ionic liquids (RTILs)1 have been recently identified as useful solvents and additives for organic synthesis, elec⇑ Corresponding author. Fax: +82 31 219 2394. E-mail address: [email protected] (T.D. Kim). Abbreviations used: RTIL, room temperature ionic liquid; [BMIM], 1-butyl-3methyl-imidazolium; ThT, thioflavin T; TEM, transmission electron microscopy; a-lac, a-lactalbumin; FSC, forward scattering; SSC, side scattering; HEWL, in hen egg white lysozyme. 1
0003-2697/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2011.08.007
troanalytical analysis, and chromatography [8,9]. In previous studies, several groups, including our group, showed that these organic salts can effectively promote or alter the self-assembly process of proteins [10–12]. In this report, we show that 1-butyl-3-methyl-imidazolium ([BMIM])-based ionic liquids (i.e., [BMIM] with different negative ions) can effectively trigger amyloid fibril formation. In a previous report, anion variation of cosolvents was shown to have a more dramatic effect on protein refolding than variation of the cations [13]. The thioflavin T (ThT) assay, flow cytometry, fluorescence microscopy, transmission electron microscopy (TEM), and other biochemical methods have been used to explore the effects of the addition of [BMIM]-based ionic liquids on amyloid fibril formation. In this study, apo a-lactalbumin (a-lac), a small Ca2+-binding protein, was used as a model protein [14] to monitor the aggregation in the presence of 5% [BMIM]-based RTILs. The effect of 5% (v/v) RTILs on the kinetics of amyloid fibril formation was evaluated by ThT assay [11,15]. Briefly, protein samples (1 mg/ml final concentration) with RTILs were incubated in 20 mM glycine buffer (pH 2.0) with constant shaking at room temperature [15]. To monitor amyloid fibril formation, 10-ll aliquots were removed at various time points during aggregation and mixed with 190 ll of ThT (10 lm final concentration in the same buffer). ThT fluorescence was measured at kex = 450 nm and kem = 490 nm after equilibrium using a spectrofluorometer (FP-6200, Jasco, Tokyo, Japan). As shown in Fig. 1A, high ThT readings indicated that amyloid fibrils were effectively formed. Although protein only and protein with [BMIM][(CF3SO2)2N] were ineffective, [BMIM][BF4] and [BMIM][PF6] were highly successful in amyloid formation with high ThT values. Specifically, [BMIM][BF4] produced highly ThT-positive amyloid fibrils compared with [BMIM][PF6]. Therefore, amyloid formation of a-lac was substantially affected by the chemical nature of
Notes & Tips / Anal. Biochem. 419 (2011) 354–356
355
Fig.1. Biochemical characterization of the amyloid formation of a-lac with [BMIM]-based ionic liquids. (A) ThT fluorescence as a function of time of apo a-lac alone (d) and in the presence of [BMIM][BF4] (D), [BMIM][(CF3SO2)2N] (j), or [BMIM][PF6] (s). (B) Fluorescence image of ThT-stained a-lac amyloid fibrils with [BMIM][BF4]. (C) Flow cytometric analysis of a-lac amyloid fibrils. Typical scattering pattern (FSC vs. SSC) is shown for protein only and for protein with [BMIM][(CF3SO2)2N], [BMIM][BF4], or [BMIM][PF6].
negative ions. In the absence of these ionic liquids, no detectable changes of fluorescence were observed at room temperature (Fig. 1A). The order of amyloid fibril-forming ability for a-lac was shown as BF4 > PF6 > (CF3SO2)2N , CH3SO4 . Amyloid fibrils induced by [BMIM][BF4] were clearly observable by a fluorescence microscope equipped with a 100-W mercury lamp using a BP filter of 470–500 nm (Fig. 1B). A FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) equipped with a 488-nm argon laser for forward scattering (FSC) and side scattering (SSC) was used for flow cytomet-
ric analysis. Sensitivity and gain (sensitivity = 400, gain = 20 [10 for FSC and SSC]) were optimized for the detection of large aggregates or amyloid fibrils. A linear relationship of FSC to SSC, a characteristic of amyloid fibrils [16], was clearly observed with amyloid fibrils prepared with [BMIM][BF4]. However, aggregates formed with [BMIM][(CF3SO2)2N] showed a pattern of high SSC (P200) with low FSC (6300), indicating the existence of large amorphous aggregates with no specific conformation. Interestingly, amyloid fibrils prepared with [BMIM][PF6] showed a unique distribution pattern
Fig.2. (A–C) Chemical structures of [BMIM]-based ionic liquids and TEM images of a-lac amyloid fibrils induced by [BMIM][(CF3SO2)2N] (A), [BMIM][BF4] (B), and [BMIM][PF6] (C). (D) Chemical structure of [BMIM][CH3SO4] (left) and pH-dependent TEM images of a-lac with [BMIM][CH3SO4] at 20 mM glycine (pH 2.0) (middle) and at 10 mM Tris–HCl (pH 8.0) (right). Scale bars correspond to 100 nm.
356
Notes & Tips / Anal. Biochem. 419 (2011) 354–356
with a broad range of SSC values that was different from [BMIM][BF4]. There was only a slight amount (<2.0%) of small aggregates in the absence of RTILs. Next, we compared the morphological features of [BMIM]based ionic liquid-induced amyloid fibrils using TEM (Fig. 2). Consistent with the ThT assay and flow cytometry analysis, many small aggregates with no fibrils were observed for [BMIM][(CF3SO2)2N] (Fig. 2A). For [BMIM][BF4] , amyloid fibrils of hundreds of nanometers length, but no small aggregates, were observed. Typical long straight fibrils with heights of 8–10 nm (7–12 nm in diameter) are usually seen in the presence of [BMIM][BF4] (Fig. 2B). Such fibrils have not been observed to form in the absence of ionic liquids. Interestingly, the amyloid fibrils formed with [BMIM][PF6] displayed short rod-like forms (Fig. 2C). These amyloid fibrils were very stable and resistant to heat treatment. Even incubation at 95 °C for 1 h did not produce any apparent disaggregation as determined by the ThT assay (data not shown). Furthermore, filial structures seeded with 2% parental forms displayed a very similar morphology to their initial shapes (data not shown). In the case of [BMIM][CH3SO4], only small globular aggregates were formed at pH 2.0 (20 mM glycine), whereas amyloid fibrils were clearly observed at pH 8.0 (10 mM Tris–HCl). This pH-responsive behavior was confirmed by TEM studies (Fig. 2D) and flow cytometry analysis (data not shown). Therefore, an ionic liquid, at least in the case of [BMIM][CH3SO4], could induce formation of amyloid fibrils or amorphous aggregates depending on the environmental pH. To ensure that the amyloid fibril-inducing effects of [BMIM]based ionic liquids were not dependent on the structure of a-lac, several other proteins were investigated with these ionic liquids. As shown in Fig. S1 in the supplementary material, these ionic liquids, especially [BMIM][BF4], promoted the formation of amyloid fibrils in hen egg white lysozyme (HEWL), calcium-saturated a-lac, insulin, and trypsin. In addition, it has been reported that [BMIM][(CF3SO2)2N] induced the appearance of amyloid fibrils in a-synuclein [11,15]. In general, optimizing fibril formation for different proteins will require trying different anions of [BMIM]based ionic liquids and different pH conditions. Consequently, it is clear that [BMIM]-based ionic liquids are highly effective for the preparation of amyloid fibrils. In summary, this report has presented a novel method to prepare different protein aggregates using [BMIM]-based ionic liquids as cosolvents. Furthermore, the morphology or properties of these aggregates can be modulated depending on the chemical structures of the ionic liquid or pH of the environment [17,18]. In addition, the effects of [BMIM]-based ionic liquids could be further regulated by chemical modifications or cross-linking reactions. The properties of ionic liquids presented in this study have potential uses in a broad spectrum of applications involving protein nanostructures or biopolymers.
Acknowledgments This work was supported by a Korean Research Foundation Grant funded by the Korean Government (KRF-2009-0089832) and by a research grant from Ajou University to T.D.K. K.K.K. was supported by the Korea Healthcare Technology R&D Project (A092006). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2011.08.007. References [1] J. Greenwald, R. Riek, Biology of amyloid: structure, function, and regulation, Structure 18 (2010) 1244–1260. [2] V.N. Uversky, D. Elizer, Biophysics of Parkinson’s disease: structure and aggregation of a-synuclein, Curr. Protein Pept. Sci. 10 (2009) 483–499. [3] R. Nelson, D. Eisenberg, Structural models of amyloid-like fibrils, Adv. Protein Chem. 73 (2006) 235–282. [4] F. Shewmaker, R.P. McGlinchey, R.B. Wickner, Structural insights into functional and pathological amyloid, J. Biol. Chem. 286 (2011) 16533–16540. [5] F. Chiti, C.M. Dobson, Protein misfolding, functional amyloid, and human disease, Annu. Rev. Biochem. 75 (2006) 333–366. [6] I. Cherny, E. Gazit, Amyloids: not only pathological agents but also ordered nanomaterials, Angew. Chem., Int. Ed. Engl. 47 (2008) 4062–4069. [7] J.W. Kelly, W.E. Balch, Amyloid as a natural product, J. Cell Biol. 161 (2003) 461–462. [8] M. Petkovic, K.R. Seddon, L.P. Rebelo, C. Silva Pereira, Ionic liquids: a pathway to environmental acceptability, Chem. Soc. Rev. 40 (2011) 1383–1403. [9] C. Hardacre, J.D. Holbrey, M. Nieuwenhuyzen, T.G. Youngs, Structure and solvation in ionic liquids, Acc. Chem. Res. 40 (2007) 1146–1155. [10] N. Byrne, C.A. Angell, Formation and dissolution of hen egg white lysozyme amyloid fibrils in protic ionic liquids, Chem. Commun. (2009) 1046–1048. [11] H. Hwang, H. Choi, H.K. Kim, H. Jo, T.D. Kim, Ionic liquids promote amyloid formation from a-synuclein, Anal. Biochem. 386 (2009) 293–295. [12] H.R. Kalhor, M. Kamizi, J. Akbari, A. Heydari, Inhibition of amyloid formation by ionic liquids: ionic liquids affecting intermediate oligomers, Biomacromolecules 10 (2009) 2468–2475. [13] R. Buchfink, A. Tischer, G. Patil, R. Rudolph, C. Lange, Ionic liquids as refolding additives: variation of the anion, J. Biotechnol. 150 (2010) 64–72. [14] D. Kurouski, W. Lauro, I.K. Lednev, Amyloid fibrils are ‘‘alive’’: spontaneous refolding from one polymorph to another, Chem. Commun. 46 (2010) 4249– 4251. [15] S.Y. Bae, S. Kim, H. Hwang, H.K. Kim, H.C. Yoon, J.H. Kim, S. Lee, T.D. Kim, Amyloid formation and disaggregation of a-synuclein and its tandem repeat (a-TR), Biochem. Biophys. Res. Commun. 400 (2010) 531–536. [16] J. Wall, A. Solomon, Flow cytometric characterization of amyloid fibrils, Methods Enzymol. 309 (1999) 460–466. [17] R. Kodali, R. Wetzel, Polymorphism in the intermediates and products of amyloid assembly, Curr. Opin. Struct. Biol. 17 (2007) 48–57. [18] J.S. Pedersen, C.B. Andersen, D.E. Otzen, Amyloid structure—one but not the same: the many levels of fibrillar polymorphism, FEBS J. 277 (2010) 4591– 4601.