Protein Cryoprotectant Ability of the Aqueous Zwitterionic Solution.

Protein cryopreservation is important for the long-term storage of unstable proteins. Recently, we found that N-acetylglucosaminyltransferase-V (GnT-V) can be cryopreserved in a deep freezer without temperature control using a dilute binary aqueous solution of 3-(1-(2-(2-methoxyethoxy)ethyl)imidazol-3-io)butane-1-carboxylate (OE2imC3C) [10 wt %, mole fraction of solute (x) = 7.75 × 10-3], an artificial zwitterion. However, it is unclear which solvent properties are required in these media to preserve unstable proteins, such as GnT-V. In this study, we investigated the melting phenomena and solution structure of dilute binary aqueous OE2imC3C solutions [x = 0-2.96 × 10-2 (0-30 wt %)] using differential scanning calorimetry (DSC) and Raman and Fourier transform infrared (FTIR) spectroscopies combined with molecular dynamics (MD) simulation to compare the cryoprotectant ability of OE2imC3C with two general cryoprotectants (CPAs), glycerol and dimethyl sulfoxide. DSC results indicated that aqueous OE2imC3C solutions can be melted at lower temperatures with less energy than the control CPA solution, with increasing x, primarily due to OE2imC3C having a higher content of unfrozen water molecules. Moreover, Raman and FTIR results showed that the high content of unfrozen water molecules in aqueous OE2imC3C solutions was due to the hydration around the ionic parts (the COO- group and imidazolium ring) and the OCH2CH2O segment. In addition, the MD simulation results showed that there were fewer structured water molecules around the OCH2CH2O segment than the hydration water molecules around the ionic parts. These solvent properties suggest that dilute aqueous OE2imC3C solutions are effective in preventing freezing, even in a deep freezer. Therefore, this medium has the potential to act as a novel cryoprotectant for proteins in biotechnology and biomedical fields.

An ionic liquid-assisted sample preparation method for sensitive integral-membrane proteome analysis.

Many ion channels and receptor proteins are potential targets for new drugs. However, standard methods for profiling these integral membrane proteins (IMPs) have not been fully established, especially when applied to rare and quantity-limited biological samples. We previously demonstrated that a mixture containing 1-butyl-3-methylimidazolium cyanate, an ionic liquid (IL), and NaOH (termed i-soln) is an excellent solubilizer for insoluble aggregates. In this study, we present a combined i-soln-assisted proteomic sample preparation platform (termed pTRUST), which is compatible with starting materials in the sub-microgram range, using our previously reported i-soln-based sample preparation strategy (iBOPs) and an in-StageTip technique. This novel and straightforward approach allows for the rapid solubilization and processing of a variety of IMPs from human samples to support highly sensitive mass spectrometry analysis. We also demonstrated that the performance of this technology surpasses that of conventional methods such as filter-aided sample preparation methods, FASP and i-FASP. The convenience and availability of pTRUST technology using the IL system have great potential for proteomic identification and characterization of novel drug targets and disease biology in research and clinical settings.

Effect of rehabilitation in patients undergoing hematopoietic stem cell transplantation.

Patients undergoing hematopoietic stem cell transplantation (HSCT) tend to experience decline in physical function, mental function, and quality of life (QOL) after HSCT due to low activity caused by adverse reactions to chemotherapy used in pre-transplantation treatment and post-transplant complications. Rehabilitation for HSCT patients is effective in preventing decline in physical function, reducing fatigue, and improving QOL. A combination of aerobic exercise and strength training is recommended for exercise therapy. Risk management is also important in the implementation of exercise therapy, and the exercise intensity should be determined according to the presence of anemia, low platelet counts, or post-transplant complications. On the other hand, post-transplant complications can decrease the patient's motivation and daily activity level. A multidisciplinary approach, which includes physicians and nurses, is important to achieve early discharge from the hospital and as quick a return to society as possible.

Cryostorage of unstable N-acetylglucosaminyltransferase-V by synthetic zwitterions.

We report biocompatible materials for cryostorage of unstable proteins such as cancer-related enzyme, N-acetylglucosaminyltransferase-V (GnT-V). GnT-V activity and the amount of protein after freezing were better retained in synthetic zwitterion solutions than in the glycerol solution. This study highlights the potential utility of synthetic zwitterions as novel cryoprotectants.

High-Pressure Raman Study of n-Octane up to 15 GPa.

The high-pressure (HP) phase transition and conformational change of n-octane (hereafter abbreviation as n-C8) up to 15.3 GPa were studied using Raman spectroscopy to investigate the relationship between the HP phase state and the alkyl chain length of n-alkanes. The Raman spectral analysis of n-C8 indicated that the liquid-solid transition (solidification) occurs at ∼0.9 GPa and that the corresponding transition pressure of n-alkanes depends on their density. Further pressurization at ∼4 GPa increased the population of the gauche conformer, while the solid (order)-amorphous transition occurred at ∼6 GPa along with a change in the full width at half maximum of the ruby R1 fluorescence line. The comparison of our findings with previously reported results suggested that the even-odd effect in the HP phase transition after solidification of n-alkanes appears between n-C7 and n-C8 as their HP phase transition up to ∼15 GPa was different.

Peculiar High-Pressure Phase Behavior of 1-Butyl-3-methylimidazolium Iodide.

We investigated the stability of the liquid phase of 1-butyl-3-methylimidazolium iodide (hereafter abbreviated as [C4mim][I]) up to 16.7 GPa at room temperature. We observed a peculiar phase transition behavior in the [C4mim][I] sample. In particular, a glassy state was formed at ∼1.3 GPa; however, the reddish-brown precipitate was formed probably due to concentrated I3- or I2- species that were formed above 12 GPa; [C4mim][I] showed a pressure-induced partial crystallization from the glassy state. We concluded that the conformation of [C4mim]+ is essential in iterative modulation to control the environmental formation of iodide precipitate.

Formation of α-synuclein aggregates in aqueous ethylammonium nitrate solutions.

The effect of adding ethylammonium nitrate (EAN), which is an ionic liquid (IL), on the aggregate formation of α-synuclein (α-Syn) in aqueous solution has been investigated. FTIR and Raman spectroscopy were used to investigate changes in the secondary structure of α-Syn and in the states of water molecules and EAN. The results presented here show that the addition of EAN to α-Syn causes the formation of an intermolecular ß-sheet structure in the following manner: native disordered state → polyproline II (PPII)-helix → intermolecular ß-sheet (α-Syn amyloid-like aggregates: α-SynA). Although cations and anions of EAN play roles in masking the charged side chains and PPII-helix-forming ability involved in the formation of α-SynA, water molecules are not directly related to its formation. We conclude that EAN-induced α-Syn amyloid-like aggregates form at hydrophobic associations in the middle of the molecules after masking the charged side chains at the N- and C-terminals of α-Syn.

An Ionic Liquid-Based Sample Preparation Method for Next-Stage Aggregate Proteomic Analysis.

A wide variety of proteomic methods have been applied for protein profiling of insoluble aggregates or inclusion bodies deposited in various cells or tissues. However, these are essentially optimized or modified classical protein chemistry techniques using conventional denaturing agents such as formic acid, urea, and sodium dodecyl sulfate (SDS). The use of these denaturants has several shortcomings, including limited solubilization, contamination, and restrictions on absolute sample quantity and throughput. Here, we describe an alternative proteomic sample preparation platform for widespread aggregation analysis. This approach combines two techniques, (1) the use of ionic liquid for protein solubilization and (2) the recently published microbead-based and organic-media-assisted proteolysis strategy (BOPs), into a single-tube workflow. We demonstrate that the combined approach (iBOPs) enabled the successful solubilization of heat-aggregated hen egg whites within 10 min and supported sensitive mass spectrometry (MS) analysis. The performance of the iBOPs system surpassed those of conventional detergents and chaotropes. Moreover, this technology enabled ultrasensitive proteomic characterization of protein aggregates deposited in individual Caenorhabditis elegans nematodes. We identified ubiquitin and other molecules as candidate stochastic factors whose accumulation levels varied among aging nematode individuals. The sensitivity and applicability of the present iBOPs make it especially attractive for next-stage aggregate proteomic analysis of various biological processes.

How does the flexibility of pyrrolidinium cations affect the phase behaviour of 1-alkyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide homologues under stressful conditions?

We conducted high-pressure Raman spectroscopy measurements on a series of 1-alkyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([Pyr1n][TFSI], n = 3, 4, 6 and 8) homologues that have different alkyl chain lengths, n, at room temperature. The results showed that all [Pyr1n][TFSI] samples formed a glassy state in which the glass transition pressure (pg) slightly increased with an increase in n. This tendency is similar to prior results of high-pressure glass formation of [Cnmim][TFSI], although the pgs for [Pyr1n][TFSI] are larger than those for [Cnmim][TFSI] with corresponding n by ∼0.5 GPa. We discuss the local structural changes occurring in [Pyr1n][TFSI] in view of the conformational changes of the Pyr+1n cation and TFSI- anion.

Long Periodic Structure of a Room-Temperature Ionic Liquid by High-Pressure Small-Angle X-Ray Scattering and Wide-Angle X-Ray Scattering: 1-Decyl-3-Methylimidazolium Chloride.

The Bragg reflections of 1-decyl-3-methylimidazolium chloride ([C10 mim][Cl]), a room-temperature ionic liquid, are observed in a lowly scattered wavevector (q) region using high-pressure (HP) small-angle X-ray scattering methods. The HP crystal of [C10 mim][Cl] was characterized by an extremely long periodic structure. The peak position at the lowest q (1.4 nm-1 ) was different from that of the prepeak observed in the liquid state (2.3 nm-1 ). Simultaneously, Bragg reflections at high-q were detected using HP wide-angle X-ray scattering. The longest lattice constant was estimated to be 4.3 nm using structural analysis. The crystal structure of HP differed from that of the low-temperature (LT) crystal and the LT liquid crystal. With increasing pressure, Bragg reflections in the high-q component became much broader, and were accompanied by phase transition, although those in the low-q component were observed to be relatively sharp.

Suppression and dissolution of amyloid aggregates using ionic liquids.

Amyloid aggregates are composed of protein fibrils with a dominant ß-sheet structure, are water-insoluble, and are involved in the pathogenesis of many neurodegenerative diseases. Development of pharmaceuticals to treat these diseases and the design of recovery agents for amyloid-type inclusion bodies require the successful suppression and dissolution of such aggregates. Since ionic liquids (ILs) are composed of both a cation and anion and are known to suppress protein aggregation and to dissolve water-insoluble compounds such as cellulose; they may also have potential use as suppression/dissolution agents for amyloid aggregates. In the following review, we present the suppression and dissolution effects of ILs on amyloid aggregates so far reported. The protein-IL affinity (the ability of ILs to interact with amyloid proteins) was found to be the biochemical basis for ILs' suppression of amyloid formation, and the hydrogen-bonding basicity of ILs might be the basis for their ability to dissolve amyloid aggregates. These findings present the potential of ILs to serve as novel pharmaceuticals to treat neurodegenerative diseases and as recovery agents for various amyloid aggregates.

Optical absorption and photoconductivity in iodine-excess ionic liquids: the case of 1-alkyl-3-methyl imidazolium iodides.

We investigated the optical absorption and photoconductivity of iodine-excess ionic liquids (ILs) based on 1-alkyl-3-methyl imidazolium iodide ([Cnmim][I]; n = 3, 4, and 6). The iodide concentration m was 2 ≦ m ≦ 8, which was determined by the molar fraction [Cnmim]+ : [Im]- = 1 : m. By adding iodine, an absorption edge shifted from 282 nm in the UV region to around 600 nm in the visible-light region. The optical bandgaps Eo decreased gradually from 2.3 eV to 1.9 eV with increasing m from 2 to 8. The alkyl-side chain lengths of the cations have little effect on the Eo. This experimental result was confirmed by ab initio molecular orbital calculations. The effects were reflected in the photoconductivity of the ILs, as expected. [C4mim][Im] exhibited greater photo-induced electron generation compared with [C3mim][Im] and [C6mim][Im]. The photoconductivity in both [C3mim][Im] and [C6mim][Im] increased slightly with increasing m. The trend of photoconductivity in [C4mim][Im] exhibited an N-shaped form. The highest photoconductivity 1.6 was observed in [C4mim][I8].

High-pressure glass formation of a series of 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide homologues.

We investigated the stability of the liquid phase of a series of 1-alkyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([Cnmim][TFSI]) homologues with different alkyl chain lengths for 3 ≤ n ≤ 10 at room temperature. We found that all [Cnmim][TFSI] samples (n = 3-10) formed a glassy state when pressure was applied. Intriguingly, the glass transition pressure (pg) slightly increases up to n = 5, reaches a plateau at n ≧ 8, and increases again at n = 10. This is completely different from the high-pressure glass formation of [Cnmim][BF4], where the pg decreases as n increases. We discussed the local structural changes occurring in [Cnmim][TFSI] in view of the conformational changes of the cation and anion, and small-angle X-ray scattering data. It seems that [Cnmim][TFSI] is resistant to external pressure and retains its local liquid structure by conformational adjustments of the cation and anion.

Cryopreservation of Proteins Using Ionic Liquids: A Case Study of Cytochrome c.

Aqueous ionic liquid (IL) solutions form a glassy state at 77 K over a wide concentration of ILs. They have potential as novel cryopreservation/refolding solvents for proteins. However, even if proteins in glass-forming concentrations of ILs are preserved at 77 K, the recovery of activity and the structure of the proteins after cryopreservation are still unclear. To achieve high recovery of protein activity and structure by removal of ILs after cryopreservation at 77 K, we studied the recovery of activity and structural stability after cryopreservation of bovine heart cytochrome c in aqueous solutions with ILs, including ethylammonium nitrate (EAN) and 1-butyl-3-methylimidazolium thiocyanate ([bmim][SCN]) over wide IL concentrations using UV-vis, Fourier transform infrared (FTIR), and circular dichroism (CD) spectroscopy. On the whole, although the addition of both ILs induced a decrease of activity and unfolding of the secondary structure of cytochrome c before and after cooling to 77 K, EAN, a weak denaturant, showed a reduction in protein damage (decrease of activity and unfolding of secondary structure) during the reheating process from 77 K (protection ability). In contrast, [bmim][SCN], a strong denaturant, did not have this protective ability. A remarkable result is that although the addition of both ILs caused cytochrome c denaturation, > 90% of activity and structure after cryopreservation (X > 10 mol %IL) was recovered after the removal of both ILs by dialysis. These recoveries after the removal of ILs are slightly higher than the results for dimethyl disulfide (DMSO), another cryoprotectant. The present results indicate that concentrated aqueous IL solutions have potential as one-pot (i.e., solubilization/preservation/refolding) solvents for proteins, which easily aggregate after purification, with comparable results to DMSO.

TRIM32-Cytoplasmic-Body Formation Is an ATP-Consuming Process Stimulated by HSP70 in Cells.

The spontaneous and energy-releasing reaction of protein aggregation is typically prevented by cellular quality control machinery (QC). TRIM32 is a member of the TRIM (tripartite motif-containing) ubiquitin E3 ligases, and when overexpressed in cultured cells, readily forms spherical inclusions designated as cytoplasmic bodies (CBs) even without proteasome inhibition. Here, we show that HSP70, a central QC component, is a primary binding factor of overexpressed TRIM32. Contrary to expectation, however, we find that this molecular chaperone facilitates and stabilizes CB assembly depending on intrinsic ATPase activity, rather than preventing CB formation. We also show that the HSP70-TRIM32 complex is biochemically distinct from the previously characterized 14-3-3-TRIM32 phospho-complex. Moreover, the two complexes have opposing roles, with HSP70 stimulating CB formation and 14-3-3 retaining TRIM32 in a diffuse form throughout the cytosol. Our results suggest that CB inclusion formation is actively controlled by cellular QC and requires ATP, similar to protein folding and degradation reactions.

Conformational adjustment for high-pressure glass formation of 1-alkyl-3-methylimidazolium tetrafluoroborate.

The conformational stability of 1-alkyl-3-methylimidazolium tetrafluoroborate ([Cnmim][BF4], n = 3-8) under high pressure was investigated using Raman spectroscopy to reveal the preferential role of the alkyl-chain length (n) in high-pressure glass transition. To evaluate this, we determined the intensity ratio (r) and differences in the partial molar volume (ΔVtrans→gauche) between the whole trans and gauche conformers of the [Cnmim] cation using Raman intensities. Interestingly, both values were classified into a two alkyl-chain length region at the border of n = 5. The coulombic interaction (cation-anion interaction) for the conformational stability is the predominant factor below n = 5 (the cation-head portion: alkyl carbon number C < 5), and the alkyl-chain packing effect (cation-cation interaction) is the predominant factor above n = 5 (the cation-tail portion: C > 5). In combination with the conformational preference of the [Cnmim] cation under a high-pressure glassy state, the alkyl chain displays a preferential role, i.e., an increase in the gauche conformer of [Cnmim][BF4] adjusts to avoid crystallization (the conformational adjustment effect). In the presence of the coulombic interaction, the preferential role of the flexible alkyl chain is an important key to elucidate the mechanism of the complicated high-pressure phase transition behavior of ionic liquids.

Anomalous Freezing of Nano-Confined Water in Room-Temperature Ionic Liquid 1-Butyl-3-Methylimidazolium Nitrate.

Non-crystal formation of ice is investigated by simultaneous X-ray diffraction and differential scanning calorimetry measurements upon cooling to -100 °C. At room temperature, size-tunable water confinement (≈20 Šsize) in a room-temperature ionic liquid (RTIL, 1-butyl-3-methylimidazolium nitrate, [C4 mim][NO3 ]) exists in a water-rich region (70-90 mol % D2 O). The confined water (water pocket) is characterized by almost monodispersive size distribution. In [C4 mim][NO3 ]-x mol % D2 O (70

Interaction Site between the Protein Aggregates and Thiocyanate Ion in Aqueous Solution: A Case Study of 1-Butyl-3-methylimidazolium Thiocyanate.

To investigate the interaction site between amyloid-like protein aggregates and thiocyanate (SCN(-)) ion, we studied the relationship between protein aggregation (cytochrome c, myoglobin, lysozyme, ribonuclease A, and ß-lactoglobulin) and SCN(-) ion in aqueous 1-butyl-3-methylimidazolium thiocyanate ([bmim][SCN]) solutions using optical spectroscopy. The addition of [bmim][SCN] (>10 mol % IL) to a protein solution induced protein aggregation owing to the intermolecular ß-sheet structures except in the case of cytochrome c. Analysis of the content of 20 amino acid residues for each protein revealed that the degree of intermolecular ß-sheet structures (ß%) and midpoint concentration from the unfolding to aggregation state ([IL]1/2(U →ßA)) is correlated primarily with the content of Lys residue in proteins (correlation coefficient (R(2)) = 0.97). The attractive interaction between the SCN(-) ions and NH3(+) groups of the side chain terminal of Lys residue inhibits protein aggregation owing to the intermolecular ß-sheet structure. This finding might be related to the mechanism for the solubilization of amyloid aggregates by strong denaturants containing SCN(-) ions such as guanidine thiocyanate.

Stability of the Liquid State of Imidazolium-Based Ionic Liquids under High Pressure at Room Temperature.

To understand the stability of the liquid phase of ionic liquids under high pressure, we investigated the phase behavior of a series of 1-alkyl-3-methylimidazolium tetrafluoroborate ([Cnmim][BF4]) homologues with different alkyl chain lengths for 2 ≤ n ≤ 8 up to ∼7 GPa at room temperature. The ionic liquids exhibited complicated phase behavior, which was likely due to the conformational flexibility in the alkyl chain. The present results reveal that [Cnmim][BF4] falls into superpressed state around 2-3 GPa range upon compression with an implication of multiple phase or structural transitions to ∼7 GPa. Remarkably, a characteristic nanostructural organization in ionic liquids largely diminishes at the superpressed state. The behaviors of imidazolium-based ionic liquids can be classified into, at least, three patterns: (1) pressure-induced crystallization, (2) superpressurization upon compression, and (3) decompression-induced crystallization from the superpressurized glass. Interestingly, the high-pressure phase behavior was relevant to the glass transition behavior at low temperatures and ambient pressure. As n increases, the glass transition pressure (pg) decreases (from 2.8 GPa to ∼2 GPa), and the glass transition temperature increases. The results indicate that the p-T range of the liquid phase is regulated by the alkyl chain length of [Cnmim][BF4] homologues.