Recovery and cryopreservation of insulin amyloid using ionic liquids

Accepted Manuscript Recovery and cryopreservation of insulin amyloid using ionic liquids

Yuka Ishikawa, Takahiro Takekiyo, Yukihiro Yoshimura PII: DOI: Reference:

S0167-7322(18)30687-1 doi:10.1016/j.molliq.2018.10.075 MOLLIQ 9820

To appear in:

Journal of Molecular Liquids

Received date: Revised date: Accepted date:

7 February 2018 26 August 2018 15 October 2018

Please cite this article as: Yuka Ishikawa, Takahiro Takekiyo, Yukihiro Yoshimura , Recovery and cryopreservation of insulin amyloid using ionic liquids. Molliq (2018), doi:10.1016/j.molliq.2018.10.075

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ACCEPTED MANUSCRIPT Recovery and Cryopreservation of Insulin Amyloid Using Ionic Liquids Yuka Ishikawa, Takahiro Takekiyo*, and Yukihiro Yoshimura Department of Applied Chemistry, National Defense Academy, 1-10-20, Hashirimizu, Yokosuka, Kanagawa, 239-8686 Japan.

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*Corresponding author: e-mail: [email protected]

Abstract: Amyloid aggregates containing a high content of β-sheet structure are one of several water-

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insoluble protein aggregates. In this study, we assessed the use of aqueous ionic liquid (IL) solutions

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as part of a simple preservation/refolding technique for amyloid aggregates. We investigated the dissolution and refolding of cryopreserved bovine insulin amyloid in concentrated aqueous solutions

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(X = 20 mol%IL) of six different ionic liquids: three 1-butyl-3-methylimidazolium-based ILs

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([bmim][X]; X = SCN, NO3, or Cl), 1-ethyl-3-methylimidazolium nitrate ([emim][NO3]), ethylammonium nitrate (EAN), and propylammonium nitrate (PAN). Dimethyl sulfoxide (DMSO) was used as the reference standard for spectroscopy. The six ILs all exhibited the ability to dissolve

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insulin amyloid, with imidazolium-based ILs exhibiting increased dissolution capacity compared with

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ammonium-based ILs or DMSO. Remarkably, >80 % of the secondary structure of the folded insulin monomer was recovered from insulin amyloid dissolved in aqueous IL solutions following

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cryopreservation and IL removal by dialysis. These results indicate that concentrated aqueous IL solutions have potential as a new and simple agent for the dissolution, cryopreservation, and refolding

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of amyloid aggregates.

Keywords: Insulin amyloid; Ionic liquids; Recovery; Cryopreservation; Spectroscopy

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ACCEPTED MANUSCRIPT 1. Introduction The constituent cations and anions of ionic liquids (ILs) can be varied to produce solvents with different chaotropic and kosmotropic properties. These properties are critical in determining the aqueous solubility and protein stability of the solute. Recently, ILs are widely used in bioscience and biomaterial applications such as protein storage, buffer preparation, and biocatalysis [1-3]. An

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intriguing feature of aqueous IL solutions was demonstrated by Fujita et al.[4]; aggregated

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recombinant CcCel6A protein (52.2 kDa) expressed in Escherichia coli (E. coli) could be solubilized

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with concentrated ILs such as choline dihydrogen phosphate ([Chl][dhp]). Previous differential thermal analysis (DTA) and Raman spectroscopic studies have shown that concentrated aqueous IL

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solutions easily form a glassy state at 77 K over a wide concentration range of X (mol%IL) = 10–100 (the concentration at which water and IL crystals do not exist, is at X ≥20) [5,6]. This glass-formation

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ability of concentrated aqueous IL solutions facilitates the cryopreservation of proteins [7]. Aqueous IL solutions may therefore have the potential to increase the solubilization of protein

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aggregates and to increase the cryopreservation of solubilized proteins. The long history of

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preservation agents that also dissolve recombinant proteins stems from the import and export of valuable proteins, and the challenges of reduced protein function resulting from the preparation of

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large-scale stock solutions. Here we focused on insulin amyloid to assess the potential of aqueous IL solutions as a preservation/refolding agent for various protein aggregates.

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Amyloid aggregates with high cross-β-sheet content are known to form through the ordered aggregation of misfolded proteins. Such aggregates are involved in many neurodegenerative diseases such as Parkinson’s disease, and are known to be transported to amyloid-type inclusion bodies in response to the overexpression of recombinant proteins [8-10]. The dissolution/preservation/refolding of amyloids is fundamental to the development of novel biomedical treatments for neurodegenerative diseases and the development of prolonged preservation techniques for recombinant proteins from amyloid-type inclusion bodies [8-11]. Although dimethyl sulfoxide (DMSO) is known to dissolve 2

ACCEPTED MANUSCRIPT amyloids [12,13] and offer cryoprotective effects [14,15], a high concentration (>80 %) of DMSO is needed to produce results [10]. In addition, concentrated DMSO solutions often induce protein aggregation and damage during the process of heating the molecules after cryogenic storage [5,16]. The development of a novel agent with high dissolution, refolding, and cryoprotectant ability, which also inhibits reaggregation, would be greatly beneficial to the industrial application of valuable

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recombinant proteins in the protein engineering and biomedical science fields. This is especially true

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for the widely encountered amyloids.

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In this study, bovine insulin amyloid was used as a model amyloid to investigate the effects of six different IL aqueous solutions (X = 20 mol%IL) on dissolution and refolding subsequent to

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cryopreservation. FTIR, UV-Vis, and circular dichroism (CD) spectroscopy techniques were used for the analyses. For the six ILs used in this study, we first selected four imidazolium-based ILs: 1-butyl-

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3-methylimidazolium-based ILs with SCN, NO3, and Cl anions ([bmim][SCN]), [bmim][NO3], and [bmim][Cl]), and 1-ethyl-3-methylimidazolium nitrate ([emim][NO3]). In addition to the imidazolium-

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based ILs, we also tested ethylammonium nitrate (EAN) and propylammonium nitrate (PAN), which

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are also known to promote helix formation [17]. The present finding is that the dissolution/cryopreservation/refolding of insulin amyloid was

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possible using concentrated IL solutions and that >80 % of the insulin was refolded after only one

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treatment with certain ILs.

2. Experimental Section 2.1. Samples.

Bovine insulin (Sigma-Aldrich) was used without further purification. [bmim][Cl] and [bmim][SCN] (Kanto Chemical Co.), [bmim][NO3] (Sigma-Aldrich), [emim][NO3], PAN and EAN (IoLiTec GmbH, Heilbronn, Germany), were all used as ILs without further purification. DMSO (Wako junyaku Co.), which is known to dissolve amyloids and cryoprotect biomolecules, was used as 3

ACCEPTED MANUSCRIPT the reference standard without further purification. Congo Red (CR) solution was purchased from Sigma-Aldrich. All mixtures containing ILs or DMSO were prepared by mixing the required amount of each in heavy water (99.9 % D2O; Sigma-Aldrich) at X = 20 mol%IL or mol%DMSO showing the glass-forming concentration [5,7]. The pD values of all aqueous solutions of the ILs and DMSO used in this study were between 3.5 and 7.2. The pD values were estimated by adding 0.4 units to the

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solutions’ pH values [18]. All samples were prepared in a dry box to avoid contamination with

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atmospheric H2O and CO2. For UV-Vis measurements, the protein concentration in each solution was

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adjusted to 1 mg/mL from a stock solution of 10 mg/mL. For the FTIR measurements, the concentrations of the insulin solutions were adjusted to 10 mg/mL with a deuterated aqueous solution

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of the ILs or DMSO. The dissolution and refolding experiments were replicated multiple times.

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2.2. Insulin amyloid.

Insulin amyloids were prepared at acidic pD and high temperature [19]. The insulin was dissolved at 10 mg/mL in 50 mM DCl (pD 1.6) containing 500 mM NaCl, after which 300 μL aliquots were

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transferred to 1.5-mL tubes and heated to 90 °C for 45 min. The solutions were removed from the heating block and water-cooled to room temperature with moderate mixing. The resulting insulin amyloid was dissolved in different aqueous solutions of the ILs or DMSO were mixed overnight at

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room temperature with a vortex. After mixing, the sample supernatants were obtained by centrifugation (4,000 rcf) for 5 min. The formation of insulin amyloid was confirmed with CR assays and FTIR

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spectroscopy. We were unable to observe the UV-Vis spectrum of the insulin amyloids dissolved in IL solutions because of the strong peak contributed by the imidazolium and alkylammonium cations [20]. Dialysis was performed with the Float A-lyzer G2 dialysis kit with a molecular weight cut-off of 3.5– 5 kD (Funakoshi Co., Ltd.). Dialysis was performed over 8 h (dialysate exchange every 2 h) using distilled water, providing a 10- to 20-fold dilution of the insulin-IL solutions into 20 mM DCl (pD 4

ACCEPTED MANUSCRIPT 1.6), without taking into account the protein lost to insulin aggregation and the dissolution of the cellulose membranes. 2.3. CR assay. The insulin solutions with and without ILs or DMSO were mixed with 12 μM CR in 50 mM

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phosphate buffer at room temperature. The sample absorption spectra were then measured over 380– 600 nm with a Genesys 10S UV-Vis spectrophotometer (Thermo Fisher Scientific) and a quartz cell

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with a 1-cm path length. Typical spectra were accumulated at a scan rate of 20 nm/min at integrals of

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0.5 nm.

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2.4. FTIR and CD spectral measurements.

FTIR spectra were recorded with a Nicolet 6700 FTIR spectrometer equipped with an MCT liquid

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nitrogen-cooled detector. Typically, 512 interferograms were collected to obtain a spectrum with a resolution of 4 cm−1. For spectral measurements, each sample was loaded into a cell with CaF2

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windows and a 50-μm Teflon spacer. All exchangeable backbone amide protons were deuterated by

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incubating the protein in a DCl solution for 1 day. Completion of the hydrogen-deuterium exchange was confirmed when the amide II band ceased to shift. This band, in the frequency region around 1550

protons.

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cm1, is known to shift to approximately 1450 cm1 following deuteration of the backbone amide

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CD spectra of the dissolved insulin amyloid solutions after dialysis were measured over 200–300 nm with a JASCO J-820 spectropolarimeter. Typical spectra were accumulated at a scan rate of 20 nm/min at integrals of 0.1 nm. The results of five scans were averaged for each spectrum. The obtained spectra were converted to molar ellipticity using [θ] = θobs/(10ncl), where θobs is the observed ellipticity, l is the cuvette path length in cm, c is the molar concentration of the proteins, and n is the number of protein residues. Solvent-only spectra were also measured under the same conditions used for the

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ACCEPTED MANUSCRIPT protein solution measurements, and these spectra were then subtracted from the protein solution spectra.

3. Results and Discussion 3.1. Dissolution of insulin amyloid using aqueous ionic liquids solutions.

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We first investigated the dissolution of insulin amyloid in six different ILs at X = 20 (the complete glass-forming concentration) at 77 K. FTIR spectroscopy and the CR assay were used to determine the

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level of dissolution. Fig. 1(a) shows the FTIR spectra in the amide Iʹ region of the insulin amyloid in

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aqueous solutions containing each of the six ILs, or DMSO or water only. The decrease in absorbance

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at 1623 cm−1 (Abs1623 cm−1) of the intermolecular β-sheet structure of the insulin amyloid [21] indicated that dissolution of the amyloid was achieved by the addition of the six ILs. The decreases in Abs1623 observed for the aqueous imidazolium-based IL solutions were larger than the decreases observed

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cm–1

for aqueous solutions containing EAN or PAN. Similar results were also obtained with the CR assay,

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as shown in Fig. 1(b). In the CR assay, absorption at 541 nm (Abs541nm) arises from CR binding with

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the amyloid [22]. A decrease in Abs541nm indicates the dissolution of the amyloid, which was observed for all six ILs at X = 20. The FTIR and CR assay results therefore demonstrated the ability of the ILs

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to dissolve insulin amyloid.

We next investigated the change in secondary structure of the insulin amyloid by applying the

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curve-fitting method to the FTIR spectra (Supporting Information). The four bands typically observed at 1623, 1640, 1654, and 1678 cm−1 in the FTIR spectra correspond to the intermolecular β-sheet structure, disordered structure, α-helical structure, and turn structure, respectively [23, 24]. Using the curve-fitted results, we could determine the insulin amyloid content in the various solutions with the following equation: Amyloid content (%) =

Ainter.β-sheet Ainter.β-sheet +Aα-helix +Adisorderd +Aturn

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,

ACCEPTED MANUSCRIPT where the value of AY represents the integrated intensity of each secondary structure (Y) determined by the curve-fitted analysis (Fig. 1(c)). The insulin amyloid levels in the imidazolium-based ILs were reduced compared with the ammonium-based ILs. Remarkably, the amyloid contents in [bmim][Cl] and [bmim][NO3] were <10 % of that in the control (water only). The rank order of dissolution ability of ILs for insulin amyloid was [bmim][Cl] = [bmim][NO3] ≥ [bmim][SCN] = [emim][NO3] ≥ DMSO

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= EAN = PAN. A similar rank order was obtained with the CR assay (based on the change in Abs541nm),

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as shown in Fig. 1(d). Although the SCN anion is a stronger denaturant than the NO3 and Cl anions,

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the rank order of this anion in our study was not consistent with the denaturant effect of an anionic species. On the other hand, the imidazolium-based cation provided increased dissolution of insulin

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amyloid compared with the alkylammonium cation ([bmim] cation > [emim] cation > propylammonium cation > ethylammonium cation). These results indicated that the dissolution of

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insulin amyloid was strongly dependent on the cationic species of the ILs tested rather the anionic species.

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Another intriguing result was that the insulin amyloid dissolved upon the addition of the ILs at X =

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20 induced an increase in absorbance at 1654 cm−1, which indicated an increase in α-helical structure (Supporting Information (b)). Fig. 2 shows the α-helical content of the dissolved insulin amyloid in

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aqueous solutions containing different ILs or DMSO only. The helical content of the dissolved insulin amyloid at X = 20 compared with that of the monomeric insulin (75%) was >65% for the imidazolium-

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based ILs, and 30–45% for the ammonium-based ILs. Helix formation in concentrated aqueous IL solutions has already been studied for proteins such as chicken lysozyme, β-lactoglobulin, and ribonuclease A, and is related to solution properties such as low polarity [17, 25, 26]. As is the case with alcohol-based denaturation [12, 27, 28], low polarity is known to weaken the hydrophobic interactions that stabilize the compact folded structure of proteins but simultaneously strengthen electrostatic interactions such as hydrogen bonds (H-bonds). This stabilizes secondary structures and in particular, the α-helical content. ILs with low polarity also enhance the intramolecular H-bonds of 7

ACCEPTED MANUSCRIPT proteins through the removal of water molecules in proximity to the protein molecular structure [29, 30]. The increase observed in the α-helical content of the dissolved insulin amyloid at X = 20 may therefore be related to the promotion of helix formation by the concentrated IL solutions; i.e., the addition of concentrated IL solutions to insulin amyloid led to the formation of α-helical structure

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through the disruption of the amyloid structure.

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3.2. Refolding of insulin structure from its amyloid form.

We next evaluated the refolding of the monomer insulin structure from its amyloid form following

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cryopreservation. In a previous study, we demonstrated the cryopreservation/refolding ability of ILs

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using cytochrome c, and showed a >90 % recovery of protein activity and structure after the removal of the ILs by dialysis [31]. Insulin amyloid dissolved in solutions of either ILs or DMSO was directly

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immersed into liquid nitrogen and kept at 77 K for ~30 min, after which the solutions were allowed to equilibrate to room temperature. We then removed the ILs using dialysis.

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As a representative result, Fig. 3(a) shows the absorption spectra of the dissolved insulin in an

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aqueous [bmim][SCN] solution at X = 20 before and after dialysis, compared with aqueous solutions of insulin or [bmim][SCN] only. A strong peak was observed between 300–350 nm resulting from the removal of [bmim][SCN] by dialysis [20]. The absorption spectra of the dissolved insulin after dialysis

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was in agreement with that of the monomer insulin. Similar absorption spectra were also obtained with

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the other ILs and DMSO at X = 20. These results indicated that the six ILs and DMSO could be effectively removed from the dissolved insulin amyloid solutions by dialysis. Moreover, CR assays of the insulin solution after dialysis showed that the insulin amyloid absorption peak at 541 nm was absent for all solutions prepared with the six ILs or DMSO as shown in Fig. 3(b). We next used far-UV CD spectroscopy to investigate the refolding of the secondary insulin structure in the dissolved and dialzyed insulin amyloid solutions. Fig. 4(a) shows the representative far-UV CD spectra of the dissolved insulin amyloid in aqueous [bmim][Cl] after dialysis compared with the 8

ACCEPTED MANUSCRIPT spectra for the monomer insulin and insulin amyloid alone. The far-UV CD spectrum of the dissolved and dialyzed insulin was very similar to that of the monomer insulin (folded state) with negative CD bands at 208 nm and 222 nm, which indicated the presence of α-helical structure rather than insulin amyloid structure (215 nm) [24]. The refolded α-helical insulin content determined by far-UV CD spectra for all six ILs and DMSO is summarized in Fig. 4(b). Overall, the α-helical content of the

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refolded insulin was >80 % of that observed for the monomer insulin. Remarkably, >90 % of the

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secondary structure of insulin was recovered from the amyloid form following removal of [bmim][Cl]

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and [bmim][NO3] and this result was close to that observed for DMSO, which has known cryoprotectant ability. These results imply that the IL additives have a positive effect on the refolding

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process. In view of the formation of a secondary structure, the present results suggest that the dissolved insulin amyloid can regenerate to the folded insulin structure via dialysis. However, whether the

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tertiary structure of dissolved insulin refolds is still unclear. Unfortunately, we could not observe the near-UVCD spectra indicating the tertiary structure because the insulin concentration obtained after

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dialysis does not allow for this measurement. Thus, it is difficult to confirm that the recovered insulin

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is in the completely native state in the present results. The combination of effective amyloid dissolution (Fig. 1(b)) and insulin refolding (based on

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refolded α-helical content; Fig. 4(b)) indicates that ILs have potential as a one-pot preservation/recovery agent for insulin amyloid, with [bmim][Cl] offering the best results among

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DMSO and the six ILs tested. One of our previous studies showed that concentrated [bmim][Cl] solutions do not exhibit low-temperature crystallization from the glassy state during the heating process [32]. Because the inhibition of low-temperature crystallization during the heating process reduces protein damage through ice nucleation, concentrated aqueous [bmim][Cl] solutions are highly conducive to cryopreservation. Finally, we mention the mechanism of insulin amyloid refolding using ILs. ILs with high dissolution ability (Fig. 1(a)) seem to induce high formation of refolded α-helical insulin (Fig. 4). The amyloid 9

ACCEPTED MANUSCRIPT content showed good correlation with the refolded α-helical insulin content, as shown in Fig. 5. This indicates that the refolding ability of ILs is directly related to their dissolution ability. Recently, we investigated the dissociation ability of concentrated IL solutions for insulin amyloid using Kamlet– Taft (KT) parameters [33]. These are a set of factors reflecting solvent properties used to describe hydrogen-bonding (H-bonding) acidity (α), basicity (β), and polarizability (π*) [34]. The β-value of

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the ILs mainly correlates with the dissolution ability of insulin amyloid in concentrated ILs solutions

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(the β-value of [bmim][Cl] is the highest among the six ILs studied) [33]. Based on these results, the

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addition of concentrated IL solutions to insulin amyloid induced the disruption of intermolecular Hbonding due to the H-bonding basicity of ILs. Additionally, dissolved insulin–IL interactions, such as

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electrostatic interaction and H-bonding, suppressed amyloid reformation [35, 36]. Moreover, because the low polarity and nano-heterogeneity structure/water hydration of aqueous IL solutions enhance

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intramolecular H-bonding in proteins, insulin dissolved in concentrated IL solutions forms a helical structure, which is close to its structure in the folded state [25, 26]. We suggest that this dissolved

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insulin state undergoes refolding via dialysis.

aqueous

ILs

solutions

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Based on these results, the dissolution, cryopreservation, and refolding of insulin amyloid using was

successful.

As

mentioned

in

the

Introduction,

the

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dissolution/cryopreservation/refolding of amyloid aggregates is fundamental to the development of novel biomedical treatments for neurodegenerative diseases, and the prolongation of preservation

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techniques for recombinant proteins from amyloid-type inclusion bodies. The recovery of active proteins from amyloid aggregates and amyloid-type inclusion bodies using a dissolution agent and refolding buffer combined with various refolding methods such as dilution, dialysis, column refolding, and microfluidic chips has improved [10]; however, methods for improving the currently timeconsuming preservation of the recovered proteins have remained elusive. The present study demonstrated the dissolution/cryopreservation/refolding of insulin amyloid using ILs with known 10

ACCEPTED MANUSCRIPT dissolution and cryoprotectant abilities. In addition, ILs with low vapor pressure can be removed from aqueous solutions using a vacuum and the sample can later be recondensed. In summary, the dissolution/cryopreservation/refolding of bovine insulin amyloids in concentrated aqueous solutions containing six different ILs was investigated using FTIR, UV-Vis, and CD spectroscopy. FTIR and CR assay results showed that ILs have the ability to dissolve insulin amyloid,

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and that imidazolium-based ILs have greater dissolution ability than ammonium-based ILs or DMSO.

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One remarkable result was the recovery of >80 % of the secondary structure of the dissolved insulin

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amyloid following dissolution in aqueous solutions of the six ILs after cryopreservation and dialysis. In particular, >90 % of the insulin secondary structure was recovered after only one treatment with

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[bmim][Cl]. We are currently planning to do the microscope and near-UVCD spectroscopic experiments to elucidate the complete dissociation/refolding of amyloid aggregates using ILs. We

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propose that ILs will prove indispensable in developing new, one-pot preservation/refolding agents for

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various amyloid aggregates.

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ACCEPTED MANUSCRIPT References

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[1] H. Weingärtner, C. Cabrele, C. Herrman, Phys. Chem. Chem. Phys. 14 (2012) 415–426. [2] T.L. Greaves, C.J. Drummond, Chem. Rev. 115 (2015) 11379–11448. [3] A.A. Tietze, F. Bordusa, R. Giernoth, D. Imhof, T. Lenzer, C. Mrestani-Klaus, I. Neudorf, K. Oum, D. Reith, A. Stark, ChemPhysChem 14 (2013) 4044–4064. [4] K. Fujita, M. Kajiyama, Y. Liu, N. Nakamura, H. Ohno, Chem. Comm. 52 (2016) 13491–13494. [5] Y. Yoshimura, T. Takekiyo, T. Mori, Chem. Phys. Lett. 664 (2016) 44–49. [6] Y. Yoshimura, H. Kimura, C. Okamoto, T. Miyashita, Y. Imai, H. Abe, J. Chem. Thermodyn. 43 (2011) 410–412. [7] C.A. Angel, Science 267 (1995) 1924-1935.

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[8] A. Signgh, A.K. Panda, J. Biosci. Bioeng. 99 (2005) 303–310. [9] N.S. De Groot, R. Sabate, S. Ventura, Trends. Biochem. Sci. 34 (2009) 408–416. [10] A. Signgh, V. Upadhyay, A.K. Upadhyay, S.M. Singh, A.K. Panda, Microb. Cell Fact. 14 (2015) 41 (pp. 1–10).

D

MA

NU

[11] T. Arakawa, S.J. Prestrelski, W.C. Kenney, J.F. Carpenter, Adv. Drug Deliv. Rev. 46 (2001) 307– 326. [12] N. Hirota-Nakaoka, K. Hasegawa, H. Naiki, Y. Goto, J. Biochem. 134 (2003) 159–164. [13] F. Meersman, C.M. Dobson, Biochim. Biophys. Acta 1764 (2006) 452–460. [14] Z-.W. Yu, P.J. Quinn, Biosci. Rep. 14 (1994) 259–281. [15] P. Huang, A. Dong, W.S. Caughey, J. Pharm. Sci. 84 (1995) 387–392.

AC

CE

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[16] M. Jackson, H.H. Mantsch, Biochim. Biophys. Acta 1078 (1991) 231–235. [17] T. Takekiyo, K. Yoshida, Y. Funahashi, S. Nagata, H. Abe, T. Yamaguchi, Y. Yoshimura, J. Mol. Liquids 243 (2017) 584–590. [18] A. Krҿżl, W. Bal, J. Inorg. Biochem. 98 (2004) 161–166. [19] E. Chatani, H. Imamura, N. Yamamoto, M. Kato, J. Biol. Chem. 289 (2014) 10399–10410. [20] A. Paul, P.K. Mandal, A. Samata, J. Phys. Chem. B 109 (2005) 9148–9153. [21] W. Dzwolak, V. Smirnovas, R. Jansen, R. Winter, Protein Sci. 13 (2004) 1927–1932. [22] W.E. Klunk, J.W. Pettegrew, D.J. Abraham, J. Histochem. Cytochem. 37 (1989) 1273–1281. [23] M. Bouchard, J. Zurdo, E.J. Nettleton, C.M. Dobson, C.L. Robinson, Protein Sci. 9 (2000) 1960– 1967. [24] J. Zurdo, J.I. Guijarro, C.M. Dobson, J. Am. Chem. Soc. 123 (2001) 8141–8142. [25] T. Takekiyo, K. Yamazaki, E. Yamaguchi, H. Abe, Y. Yoshimura, J. Phys. Chem. B 116 (2012) 11092–11097. [26] T. Takekiyo, Y. Koyama, K. Yamazaki, H. Abe, Y. Yoshimura, J. Phys. Chem. B 117 (2013) 10142–10148. [27] K. Shiraki, K. Nishikawa, K.Y. Goto, J. Mol. Biol. 245 (1995) 180–194. 12

ACCEPTED MANUSCRIPT [28] N. Hirota, K. Mizuno, Y. Goto, J. Mol. Biol. 275 (1998) 365–378. [29] V.N. Uversky, N.V. Narizhneva, S.O. Kirschstein, S. Winter, S. Löber, Fold. Des. 2 (1997) 163–172. [30] Y. Liu, D.W. Bolen, Biochemistry 34 (1995) 12884–12891.

PT

[31] T. Takekiyo, Y. Ishikawa, Y. Yoshimura, J. Phys. Chem. B, 121 (2017) 7614–7620. [32] N. Hatano, E. Yamaguchi, K. Yamazaki, H. Abe, T. Takekiyo, Y. Yoshimura, Cryobiol. Cryotechnol. 57 (2011) 125–129 (in Japanese). [33] T. Takekiyo, Y. Ishikawa, E. Yamaguchi, N. Yamada, Y. Yoshimura, submitted. [34] M.J. Kamlet, J. Luis, M. Abboud, M.H. Abraham, R.W. Taft, J. Org. Chem. 48 (1983) 2877– 2887.

AC

CE

PT E

D

MA

NU

SC

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[35] T. Takekiyo, T. Takekiyo, E. Yamaguchi, H. Abe, Y. Yoshimura, ACS Sustain. Chem. Eng. 4 (2016) 422–428. [36] T. Takekiyo E. Yamaguchi, K. Yoshida, M. Kato, T. Yamaguchi T, Yoshimura, J. Phys. Chem. B, 119 (2015) 6536–6544.

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ACCEPTED MANUSCRIPT Figure Captions Fig. 1. (a) Fourier-transform infrared spectroscopy (FTIR) spectra in the amide Iʹ region and (b) absorption spectra of CR assay of insulin amyloid in aqueous ILs solutions at X = 20. (c) Amyloid contents (%) and (d) the values of absorbance at 541 nm of insulin amyloids in aqueous solutions with ILs and DMSO at X = 20 combined with the results in water.

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Fig. 2. α-Helical contents (%) of dissolved insulin amyloids in aqueous solutions with ILs and DMSO at X = 20 combined with the results in monomer insulin and in water (X = 0).

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Fig. 3. (a) Representative absorption spectra of insulin in aqueous 1-butyl-3-methylimidazolium thiocyanate ([bmim][SCN]) solution (X = 20) before (blue dotted line) and after (red solid line) dialysis combined with the spectra [bmim][SCN] only (black solid line) and insulin only (black dashed line). (b) Absorption spectra of CR assay of dissolved insulin amyloid in aqueous IL solutions at X = 20 after dialysis.

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Fig. 4 (a) Far-UV circular dichroism spectra and (b) refolded α-helical contents (%) of dissolved insulin in aqueous solution with ILs and DMSO at X = 20 after cryopreservation by removing ILs and DMSO using dialysis.

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Fig. 5 Correlation between the amyloid contents (%) and refolded helical content (%) of dissolved insulin in aqueous solution with ILs. The correlation coefficient (R2) was determined using the CORREL function.

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ACCEPTED MANUSCRIPT Highlights ・Dissolution/cryopreservation/refolding of insulin amyloid in concentrated aqueous ionic liquids

(ILs) solutions were studied. ・Ionic liquids exhibited the ability to dissolve insulin amyloid. ・The imidazolium-based ILs have greater dissolution ability than ammonium-based ILs or DMSO.

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・The secondary structure of the dissolved insulin after cryopreservation was refolded after

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removing PAN by dialysis.

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Figure 1

Figure 2

Figure 3

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Figure 5