Abstract
The structural transitions of proteins in aqueous solutions of various ionic liquids (ILs) over a wide concentration range (x (mol% IL) = 0–30) were investigated using Fourier-transform infrared and near-UV circular dichroism spectroscopy combined with small-angle X-ray scattering. The proteins in the aqueous IL solutions showed two structural transition patterns: (i) the folded state → unfolded state → partial globular state (α-helical formation disrupted tertiary structure) and (ii) the folded state → unfolded state → aggregation (amyloid-like aggregation or disordered aggregation). We found that the helical formation of proteins in the condensed IL solutions was strongly related to the competition between the low polarity and denaturation effect of ions. Moreover, the amyloid-like aggregate formation correlated with the competition between the size of the confined water assemblies in the IL layer and the IL-amino acid residue interactions. On the basis of these results, we discussed the future applications of ILs, including their use as cryoprotectants for proteins and as agents for the suppression of amyloid formation.
Keywords
- protein
- aqueous ionic liquid solution
- aggregation
- helix formation
- optical spectroscopy
1. Introduction
Aqueous mixtures of proteins and ionic liquids (ILs), which comprise organic cations and anions and remain in the liquid state below 373 K, are employed in protein engineering applications, such as protein storage media, biocatalysts, and buffers [1–3]. Although these applications are based on the unique solvent properties of these mixtures, such as their solubility in water and solution structure [2, 4], the detailed relationship between the proteins and aqueous IL solutions at the molecular level is unclear. Thus, to realize the protein engineering applications of ILs, numerous studies have been conducted on the structural stability and activity of proteins in aqueous IL solutions [2–8].
For instance, Lange et al. [6] demonstrated that the addition of imidazolium-based ILs (up to 4 M) to renaturation buffers caused high protein renaturation without protein aggregation, whereas addition to the folded protein induced a decrease in the structural stability. Many aqueous IL solutions are found to degrade the structural stability and activity of proteins at
Recently, intriguing phenomena such as protein refolding and aggregate formation have been observed in condensed IL solutions (
Related to these phenomena, the solvent properties of ILs, such as viscosity and dielectric constant, drastically change at a certain IL concentration [2]. These changes depend on the amount of water in the mixture. In addition, it is known that IL solutions adopt a nanoheterogeneous structure with a polar domain, i.e., the ionic parts of the cations and anions, and a nonpolar domain, i.e., the alkyl chain of the cations [14–16]. In binary solutions under water-rich conditions [17], IL-water mixtures adopt IL-aggregated structures that are surrounded by bulk water molecules; therefore, the nanoheterogeneity of these systems is relatively low. However, under IL-rich conditions wherein the mixtures exhibit molten-salt-like behavior, the water molecules are scattered in the polar domain and self-assemble in the ILs. The water molecules in this state are termed “confined water” [18, 19] and the nanoheterogeneity of these systems is higher. As mentioned earlier, these solvent properties may contribute to unique structural transitions of the proteins. Thus, detailed information on protein stability over a wide range of IL concentrations is valuable, and this information would facilitate the use of ILs in protein engineering applications.
This manuscript aims to determine the structural stabilities of various model proteins over a wide concentration range of ILs using optical spectroscopy combined with small-angle X-ray scattering (SAXS). The origin of the structural transitions of proteins in condensed aqueous IL solutions has been discussed.
2. Experimental methodology
2.1. Materials
Chicken lysozyme, bovine ribonuclease A (RNase A) and bovine β-lactoglobulin (β-LG), horse cytochrome
2.2. FTIR spectroscopy
The amide I′ vibrational mode (deuterated peptide groups) in FTIR spectra is highly sensitive to the secondary structure of proteins, and thus it serves as an indicator of α-helix and β-sheet formation [20]. FTIR spectra were recorded using a Nicolet 6700 FTIR spectrometer equipped with a mercury-cadmium-telluride liquid-nitrogen detector. Typically, 512 interferograms were collected to obtain spectra with a resolution of 4 cm−1. Solvent spectra were also measured under the same conditions as those used for the protein solution measurements and were subtracted from the protein solution spectra.
2.3. CD spectroscopy
Near-UV CD spectra in the range of 250–300 nm are sensitive to the presence of specific rigid packing interactions between aromatic side chains, indicating changes in the tertiary structure [21]. CD spectra were measured over a wavelength range of 250–300 nm on a JASCO J-820 spectropolarimeter. Typically, spectra were accumulated at a scan rate of 20 nm min−1 in 0.1 nm steps. Five scans were averaged for each spectrum. The obtained spectra were converted into mean residue ellipticity units using [
2.4. SAXS
SAXS is a powerful technique for investigating protein size and the presence of protein aggregation [22]. SAXS experiments were conducted using a Kratky camera system (BioSAXS-1000, Rigaku Co.) at a brilliance of 56.0 kW mm−2. CuKα radiation (
3. Results and discussion
3.1. Structural transition of proteins in aqueous solutions with [bmim]-based ILs
As representative results, Figure 1a and b shows the FTIR amide I’ spectra of myoglobin and cytochrome
To further investigate the changes in the secondary structure of both proteins, we plotted the changes in the maximum absorbance (Abs) values of these two proteins against the [bmim][NO3] concentrations, as shown in Figure 1c and d. For myoglobin, the first decrease in the Abs value, indicating myoglobin unfolding, is observed in the region
Conversely, a drastic decrease in Abs for cytochrome
Figure 2a shows a Guinier plot of cytochrome
where
A similar result is also obtained using Kratky plots (Figure 2b). Kratky plots provide insight into the compactness of a protein, i.e., a bell shape in the plot indicates a globular protein, whereas a plateau, seen in the high
Next, we measured the changes in the tertiary structure of both proteins induced by [bmim][NO3] using near-UV CD spectroscopy (Figure 3a and b). Although the negative CD intensity at 290 nm for myoglobin and 288 nm for cytochrome
The results from the FTIR, SAXS, and near-UV CD analyses show that aqueous [bmim][NO3] solutions of up to
The most remarkable result is that condensed solutions with [bmim]-based ILs cause the formation of an α-helical structure, and intermolecular β-sheets or disordered-rich aggregation. From the previous results, the former state is similar to the intermediate in the on- or off-pathway for the protein folding process [29], and the latter state is similar to the amyloid structure associated with neurodegenerative conditions such as Parkinson’s disease [30, 31] and the structure of the inclusion body in expression proteins [30, 32]. Thus, it is important to reveal the origin of the structural formation of proteins in condensed aqueous IL solutions in view of protein engineering application using ILs. In the following sections, we discuss the preferential formation of the α-helical structure (PG state) in Section 3.2, and intermolecular β-sheet aggregation in Section 3.3.
3.2. Helix formation ability of ILs for proteins
We found that condensed aqueous solutions with [bmim]-based ILs induce the helical formation disrupted tertiary structure (PG state) for some proteins. Generally, it is well known that β-LG and RNase A, having substantial β-sheet contents, take non-native helical formations in aqueous alcohol solutions, such as 2,2,2-trifluoroetahnol [33–35]. This is termed alcohol denaturation. The PG state in condensed IL solutions structurally resembles that from alcohol denaturation. Here, we focused on the details of helical formation ability of ILs for β-LG and RNase A.
Figure 5a and b shows the FTIR spectra of β-LG and RNase A in aqueous [bmim][NO3] solutions of several concentrations. On the whole, the absorbance of both proteins at ca. 1635 cm−1, indicating intramolecular β-sheet structure, decreases, and that at ca. 1656 cm−1, indicating the α-helix structure, increases with [bmim][NO3] concentration (Figure 5a and c). Both proteins undergo helix formation at high [bmim][NO3] concentrations, though β-LG forms an intermolecular β-sheet structure in addition to an α-helical structure. Characterization of the helix formation in condensed aqueous [bmim][NO3] solutions reveals that it is similar to that seen for alcohol denaturation, which results in a direct β-α transition. However, it is intriguing whether the condensed [bmim][NO3] solutions induce direct β-α transition, as in the case of alcohol denaturation.
We assessed the second-derivative FTIR spectra of β-LG and RNase A in aqueous [bmim][NO3] solutions at different concentrations of [bmim][NO3] (Figure 5c and d). Increasing the [bmim][NO3] concentration up to
Here we discuss the origin of helix-forming ability of [bmim]-based ILs for β-LG and RNase A. Generally, alcohol denaturation is thought to arise from solvent properties such as low polarity. Low solvent polarity weakens the hydrophobic interactions that stabilize the compact native structure of proteins while simultaneously strengthening the intramolecular electrostatic interactions, such as hydrogen bonds, and stabilizing secondary structures, particularly the α-helix. The dielectric constant (
Next we discuss the generality of helix formation of ILs with NO3−. The helix-forming ability of [bmim][NO3] for proteins has been connected to its low polarity; however, similar solution properties are also observed in other ILs with NO3−anion. To elucidate the generality of helix formation of β-sheet-rich proteins in ILs with NO3−, we compared two imidazolium-based ILs ([bmim][NO3] and 1-ethyl-3-methylimidazolium nitrate ([emim][NO3])) and the three alkylammonium nitrates (RAN-ILs): MAN, EAN, and PAN.
As a representative result, the FTIR spectra of RNase A in condensed aqueous solutions with various ILs at
On the basis of these results, we can conclude that the helix-forming ability of IL depended on the anionic species rather than the cationic species. Besides, this ability is strongly related to the competition between the low polarity and denaturation effect of anions.
3.3. Ionic liquid-induced amyloid-like aggregation
Another intriguing phenomena associated with condensed IL solutions are the formation of intermolecular β-sheet structures (i.e., amyloid-like aggregation). As mentioned in Section 3.1, amyloid-like aggregation is related to neurodegenerative diseases such as Parkinson’s disease and the structure of the inclusion body [30–32]. The origin of amyloid-like aggregate formation in ILs is related to the suppression of protein aggregation. Consequently, we focused on amyloid-like aggregate formation in condensed aqueous solutions of [bmim][NO3] and [bmim][SCN].
First, we address the case of [bmim][NO3]. Figure 7a shows the FTIR spectra of the eight model proteins, which have different secondary structures and sizes in condensed aqueous [bmim][NO3] solutions (
Here, to investigate the influence of the imidazolium cation alkyl chain length on the decrease in amyloid-like aggregation, FTIR spectra were recorded for proteins in aqueous solutions (
In order to gain insight into the amyloid-like aggregate formation, we have focused on the solution structure and protein size. As discussed in Section 1, it has been suggested that the structural changes of proteins in aqueous IL solutions are strongly related to the solution structures of these media. 1-Alkyl-3-methylimdazolium-based ILs form nanoheterogeneous structures containing polar and nonpolar domains. The solutions exhibit molten-salt-like behavior, and the water molecules are scattered in the polar domain and self-assemble into confined-water-type domains under IL-rich conditions. The SAXS and small-angle neutron scattering (SANS) results imply that confined water exists in aqueous [bmim][NO3] solutions at
On the basis of these results, we propose that aggregated proteins in aqueous [bmim][NO3] or [emim][NO3] solutions at
Next, we discuss amyloid-like aggregation in aqueous [bmim][SCN] solutions showing the strong denaturant. Figure 8 shows the FTIR spectra of five of the investigated proteins (cytochrome
To further investigate the changes in the secondary structures of the proteins, we determined their intermolecular-β-sheet contents (β%) using curve-fitting analysis. The β% values at
To investigate the correlation between the β% values and the occurrence of 20 amino acid residues in the five investigated proteins, we determined the correlation coefficient (
Here, we can speculate that a Lys-rich polypeptide does not form an intermolecular β-sheet structure in aqueous [bmim][SCN] solutions if Lys residues are directly related to the formation of intermolecular β-sheet structures. To confirm this speculation, we measured the FTIR spectra of poly-L-lysine (PLL) (Lys = 100%) in aqueous [bmim][SCN] solutions at
These results indicate that the origin of amyloid-like aggregate formation in [bmim][NO3] is different from that in [bmim][SCN]. The former is due to the relationship between the protein size and the confined water size in the IL layer, while the latter is due to IL-amino acid residue interactions. The SCN− anion is a stronger denaturant than the NO3− anion. As the denaturation effect of the anions becomes stronger, the origin of amyloid-like aggregation in condensed IL solutions changes from solution structural properties to the IL-amino acid residue interactions.
3.4. Future application of protein engineering
We have discussed the unique structural transitions of proteins in aqueous solutions with [bmim]-based ILs. Aqueous [bmim]-based IL solutions induce two structural transition patterns: the folded state → unfolded state → intermolecular β-sheet aggregation for myoglobin, and the folded state → unfolded state → partial globular state. These transitions are strongly related to solution properties, such as the presence of confined water around the IL layers (i.e., the nanoheterogeneity), a low polarity, and IL-amino acid residue interaction. On the basis of these results, we propose that future applications of ILs in protein engineering may be as cryoprotectants for proteins and as agents for the suppression of amyloid formation.
The inhibition of ice-nucleation and a high structural reversibility of proteins without protein aggregation are important criteria for a protein cryoprotectant. Recently, Yoshimura et al. reported that the aqueous IL solutions in the wide IL concentration range exhibit glassy formation at 77 K [47]. In addition, the present study shows that condensed IL solutions cause the helical formation for some proteins without protein aggregation. Related to these results, we found that low temperatures (77 K) induce structural reversibility for lysozyme in aqueous [bmim][NO3] solutions [48]. After cooling, the lysozyme structure shows reversible transition without aggregation. Similar results were obtained in the case of RNase A in aqueous solutions of choline dihydrogen phosphate [49]. Thus, condensed IL solutions forming the glassy state at 77 K that induce helical formation without protein aggregation may be applicable as cryoprotectants for proteins, specifically as cryopreservation agents for recombinant proteins. However, in order to use ILs as cryoprotectants, it is necessary to investigate the enzyme activity in condensed IL solutions after cooling and removal of the IL from aqueous protein solutions.
As mentioned in Section 3.3, we have demonstrated that specific IL-amino acid residue interactions in condensed IL solutions cause inhibition of amyloid-like aggregation (i.e., intermolecular β-sheet structures). Related to this, we found that [bmim][SCN], EAN, and PAN ILs suppress thermally induced insulin amyloid formation [50]. Furthermore, condensed solutions of EAN or PAN demonstrate a high protectant ability for the structure of monomeric insulin. The affinity between ILs and specific amino acid residues in insulin is the main cause of the suppression of insulin amyloid formation. Thus, ILs can potentially be used as agents for the suppression of amyloid aggregation.
We proposed the applications of ILs in protein engineering as cryoprotectants for proteins and as agents for the suppression of amyloid formation using properties of the condensed IL solutions. In addition to these, the solution properties of condensed IL solution (the presence of confined water around the IL layers, a low polarity, and IL-amino acid residue interaction) will have a wide potential for applications of ILs in protein engineering in the future.
4. Conclusion
We have investigated the structural transition of proteins in aqueous solutions of ILs over a wide concentration range using FTIR and near-UV CD spectroscopy combined with SAXS. Aqueous IL solutions induced two structural transition patterns; (i) the folded state → unfolded state → partial globular state (α-helical formation disrupted tertiary structure), and (ii) the folded state → unfolded state → aggregation (amyloid-like aggregation or disordered aggregation). These transition patterns are strongly related to the condensed IL solution properties, such as the presence of confined water in the IL layers, low polarity, denaturant effect of anions, and IL-amino acid residue interactions. On the basis of these results, we proposed the new application of ILs as novel cryoprotectants and amyloid suppression agents. The present results will be basic information for the design of ILs for protein engineering applications. Although we have fully investigated the structural properties of proteins in aqueous IL solutions, detailed information on enzyme activity and methods for the removal of ILs from these media is still required to use the protein engineering applications.
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