Liquid compositions and methods for stabilizing membrane proteins - Patents.com

Abstract

To provide liquid compositions and methods for stabilization of membrane protein.SOLUTION: A liquid composition contains specific anions and specific cations. In the inventive liquid composition, preferable combinations of cations and anions are, for example: cholinium cation (Ia) and dihydrogenphosphate anion (IIa); cholinium cation (Ia) and dihydrogen citrate anion (IIb); cholinium cation (Ia) and thiocyanate anion (IIc); acetylcholine cation (Ib) and chlorine ion; 1-(2-Hydroxyethyl)-3-methylimidazolium cation (Ic) and dihydrogenphosphate anion (IIa).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to liquid compositions and methods for stabilizing membrane proteins. [Background technology]

[0002] Membrane proteins are mainly present on cell membranes and play important biological roles, making them particularly important targets in drug discovery research. However, they are difficult to handle outside of the body, limiting the scope of research. Specifically, membrane proteins are expressed in small amounts, and unlike water-soluble proteins, they have low solubility in solutions, making it difficult to obtain sufficient stability in solutions.

[0003] In order to stabilize membrane proteins isolated using surfactants or the like, the use of liposomes, lipid nanodiscs, and the like has been proposed (Non-Patent Documents 1 and 2).

[0004] However, the preparation of liposomes requires operations such as vacuum concentration and dialysis, as well as repeated freezing and thawing using liquid nitrogen, and therefore requires special techniques and several days of time. Liposomes can also be difficult to measure physicochemically depending on the size of the vesicles. Furthermore, evaluation of the efficiency of membrane protein incorporation into liposomes requires multiple steps of operations. In addition, the orientation of membrane proteins in liposomes is not always uniform, and is not easy to control.

[0005] Lipid nanodiscs are disk-shaped particles composed of proteins modified from human-derived lipoprotein A and phospholipids. The preparation of lipid nanodiscs requires many days of expression and purification of the proteins that make up the nanodiscs, and requires complicated operations and skilled techniques. In addition, the molar ratio of the disks to the phospholipids must be adjusted when reconstituting membrane proteins into nanodiscs. Furthermore, after the reconstitution operation, nanodiscs incorporating membrane proteins and empty nanodiscs that do not contain membrane proteins may be mixed, and further operations may be required to remove empty nanodiscs and / or excess membrane proteins.

[0006] As described above, there is room for improvement in the techniques for stably maintaining membrane proteins in vitro, and there is a need for a simple method for stabilizing membrane proteins as an alternative to conventional methods. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Methods Enzymol,464,211,2009 [Non-Patent Document 2] Nature commun,12,2202,2021 Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention aims to provide liquid compositions and methods for stabilizing membrane proteins. [Means for solving the problem]

[0009] The present inventors have previously reported that water-soluble proteins can be dissolved in a liquid (hereinafter referred to as "hydrated ionic liquid (Hy IL)") prepared by adding a small amount of water to an ionic liquid (IL) having a melting point of 100°C or less, and that the storage stability and thermal stability of the dissolved water-soluble protein are dramatically improved (References 1 and 2). In this study, the thermal stability of water-soluble proteins dissolved in hydrated ionic liquid was examined by observing changes in amide bands from infrared absorption spectra.

[0010] Next, the inventors investigated the application of hydrated ionic liquids to membrane proteins, which have structures and properties significantly different from those of water-soluble proteins, and surprisingly found that it is possible to dissolve membrane proteins in hydrated ionic liquids while maintaining their higher-order structures. Specifically, as a result of intensive research, the inventors found that membrane proteins can be well dissolved in hydrated ionic liquids that contain specific cations and specific anions and contain a small amount of water relative to the ion pair, while maintaining their higher-order structures, and thus completed the present invention. Furthermore, the inventors found that membrane proteins dissolved in hydrated ionic liquids have improved at least one of storage stability and thermal stability.

[0011] That is, the present invention is as follows: [1](a) The following: [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R2, R3 and R4 are hydrogen or a C1 to C4 hydrocarbon group (excluding cations in which R1, R2, R3 and R4 are all alkyl). and a cation selected from (b) The following: [ka] and an anion selected from Including, a liquid composition comprising 3 to 13 water molecules for each pair of the cation and the anion; [2] In formula (I), X is a nitrogen atom or a phosphorus atom, R1, R2 and R3 are hydrogen or C1-C4 alkyl, or R1, R2 and R3 together form a five- or six-membered ring, which may be substituted with C1-C4 alkyl; The liquid composition according to [1], wherein R4 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; [3] The cation is selected from the group consisting of: [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R2 and R3 are hydrogen or C1-C4 alkyl; R 41 is C1-C4 alkylene, R 42 is hydrogen, C1-C2 alkyl, or C1-C4 acyl. or the following: [ka] (wherein X is a nitrogen atom or a phosphorus atom, Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen and / or phosphorus atoms; R6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R7 is C1-C4 alkyl. The liquid composition according to [1] or [2], selected from the following: [4] The cation is selected from the group consisting of: [ka] (In the formula, X1 and X2 may be the same or different and are nitrogen atoms or phosphorus atoms, Ring A is a five- or six-membered heterocycle; R6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R7 is C1-C4 alkyl. The liquid composition according to [3], selected from the following: [5] The cation is selected from the group consisting of: [ka] The liquid composition according to any one of [1] to [4], selected from the following: [6] The liquid composition according to any one of [1] to [5], further comprising a membrane protein; [7] The liquid composition according to [6], wherein the membrane protein is an α-helical transmembrane protein; [8] The liquid composition according to [7], wherein the α-helical transmembrane protein has 1 to 17 transmembrane domains; [9] A method for stabilizing a membrane protein, comprising adding the membrane protein to a liquid composition containing a cation and an anion, (a) the cation is [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R 2、 R3 and R4 are hydrogen or a C1 to C4 hydrocarbon group (excluding cations in which R1, R2, R3 and R4 are all alkyl). is selected from (b) the anion is [ka] is selected from The method, wherein the liquid composition contains 3 to 13 water molecules for each pair of the cation and the anion. Effect of the Invention

[0012] According to the present invention, a liquid composition and method for stabilizing membrane proteins can be provided. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows CD spectra of TehA in buffer and in hydrated ionic liquid. [Figure 2A] FIG. 1 shows the CD spectrum change with temperature of TehA in a buffer solution. [Figure 2B] FIG. 1 shows the change in CD spectrum with temperature for TehA in a hydrated ionic liquid. [Figure 3A] FIG. 1 shows CD spectra of TehA in a buffer solution and in hydrated ionic liquids with different numbers of water molecules. [Figure 3B] FIG. 1 shows the change in CD spectrum intensity (208 nm) of TehA with temperature in a buffer solution and in hydrated ionic liquids with different numbers of water molecules. [Figure 3C] FIG. 1 shows the change in CD spectral intensity (208 nm) of TehA with temperature in buffer, [ch][dhp]-4 and Tre-30. [Figure 3D] FIG. 1 shows the change in CD spectrum intensity (208 nm) of TehA with temperature in a buffer solution and in [ch][dhp] hydrated ionic liquids with different numbers of water molecules. [Figure 3E] The denaturation temperatures of TehA in buffer and in [ch][dhp] hydrated ionic liquids with different numbers of water molecules are shown. [Figure 4A] FIG. 1 shows bR dissolved in buffer and in hydrated ionic liquid. [Figure 4B] FIG. 1 shows bR dissolved in buffer, PEG, and various hydrated ionic liquids. [Figure 4C] FIG. 1 shows bR dissolved in a buffer solution and in various hydrated ionic liquids. [Figure 5A] FIG. 1 shows the UV-visible absorption of bR in buffer and in hydrated ionic liquid. [Figure 5B] FIG. 1 shows the time course of bR incubated at 90° C. in a buffer solution and in a hydrated ionic liquid. [Figure 6]FIG. 1 shows the color change of bR in buffer and hydrated ionic liquid upon heating. [Figure 7A] FIG. 1 shows the time course of optical absorption by bR in a hydrated ionic liquid upon flash excitation. [Figure 7B] FIG. 1 shows the time course of light absorption by bR in a buffer solution upon flash excitation. [Figure 8A] FIG. 1 shows an outline of the photoreaction cycle of bR and the corresponding time constants. [Figure 8B] FIG. 1 shows the time constants in the photocycle of bR in buffer and hydrated ionic liquid. [Figure 9] FIG. 1 shows the change in UV-visible absorption of bR in hydrated ionic liquid before and after flash measurement. [Figure 10A] FIG. 1 shows the UV-visible absorbance of bR in buffer. [Figure 10B] FIG. 1 shows the UV-visible absorption of bR in [ch][dhp]-4. [Figure 10C] FIG. 1 shows the UV-visible absorption of bR in Betain-7. [Figure 11] FIG. 1 shows the thermal denaturation temperatures of bR in buffer solutions and each hydrated ionic liquid. [Figure 12A] FIG. 1 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-4. [Figure 12B] FIG. 1 shows the temperature dependence of UV-visible absorption of bR in [ch][bit]-6. [Figure 12C] FIG. 13 shows the UV-visible absorption of bR in [C2mimOH][dhp]-3. [Figure 12D] FIG. 1 shows the thermal denaturation temperatures of 50% and 80% bR in each hydrated ionic liquid. [Figure 13A] FIG. 1 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-4. [Figure 13B] FIG. 1 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-7. [Figure 13C] FIG. 13 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-15. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The following is a detailed description of the embodiments of the present invention. The present invention is not limited to the following embodiments, and can be modified in various ways within the scope of the present invention.

[0015] The present invention is based on the finding that membrane proteins can be dissolved while maintaining their higher-order structure in a hydrated ionic liquid containing a specific cation and a specific anion. Here, since "ionic liquid (IL)" has the characteristics of being non-volatile and flame-retardant and being liquid over a wide temperature range, it is possible to use it as a solvent for biomolecules. However, it is difficult to dissolve biomolecules in commonly used ionic liquids, and even if they are dissolved, the higher-order structure changes significantly. In addition, it is necessary to modify the surface of the biomolecules with polymers or the like, or to use additives. On the other hand, "hydrated ionic liquid (Hy IL)" is a mixture of an ionic liquid (IL) whose ionic structure is selected in consideration of the affinity with biomolecules and a small amount of water, and the water present in the hydrated ionic liquid is not free water present in an aqueous solution, but exists as bound water interacting with ions. "Hy IL" refers to an ionic liquid-water mixture in which no free water exists. In this specification, the term "higher-order structure" refers to a secondary structure such as an α-helix and a β-strand, and a structure of a higher dimension than the secondary structure, and may include a tertiary structure and / or a quaternary structure in addition to the secondary structure. The term "higher-order structure" includes the term "tertiary structure." In this specification, the terms "tertiary structure," "three-dimensional structure," and "steric structure" are synonymous and are used interchangeably.

[0016] The first aspect of the present invention comprises: (a) The following: [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R2, R3 and R4 are hydrogen or a C1 to C4 hydrocarbon group (excluding cations in which R1, R2, R3 and R4 are all alkyl). and a cation selected from (b) The following: [ka] and an anion selected from The present invention relates to a liquid composition comprising:

[0017] The liquid composition according to the present invention further contains, for example, 3 to 13 water molecules per pair of cation and anion (hereinafter also referred to as "one ion pair"). If the number of water molecules is too small, the ionic liquid will not become completely liquid at room temperature, while if the number of water molecules is too large, the structure characteristic of membrane proteins may not be maintained. In addition, if the number of water molecules is too large, it becomes more difficult to obtain thermal stability as the number of water molecules increases. In one embodiment, the number of water molecules contained is 4 to 13 per one ion pair. In another embodiment, the number of water molecules contained is 3 to 10 per one ion pair. In another embodiment, the number of water molecules contained is 3 to 7 per one ion pair.

[0018] The liquid composition according to the present invention can stabilize membrane proteins by containing a small number of water molecules, a specific cation, and a specific anion. Therefore, the liquid composition according to the present invention may further contain a membrane protein. Since the liquid composition according to the present invention uses an organic salt as the cation and / or anion, the interaction between ions is small and the liquid composition has a structure designability, so that the solvent properties can be more easily controlled than conventional solvents such as water and organic solvents.

[0019] In one embodiment, the cation is (a) [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R2 and R3 are hydrogen or C1-C4 alkyl, or R1, R2 and R3 together form a five- or six-membered ring, which may be substituted with C1-C4 alkyl; R4 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain. is selected from.

[0020] In another embodiment, the cation is: [ka] (wherein X is a nitrogen atom or a phosphorus atom, R1, R2 and R3 are hydrogen or C1-C4 alkyl; R 41 is C1-C4 alkylene, R 42 is hydrogen, C1-C2 alkyl, or C1-C4 acyl. is selected from.

[0021] In formula (I-1), R1, R2 and R3 are preferably hydrogen or C1 to C2 alkyl, and more preferably C1 to C2 alkyl.

[0022] In the formula (I-1), preferably: R 41 is C1-C2 alkylene, R 42 is C1-C2 alkyl or C1-C4 acyl, or R 41 is C1-C4 alkylene, R 42 is hydrogen, More preferably: R 41 is C1-C2 alkylene, R 42 is C1-C2 alkyl or C1-C2 acyl, or R 41 is C2-C4 alkylene, R 42 is hydrogen, Even better: R 41is C1-C2 alkylene, R 42 is acetyl or methyl, or R 41 is a C2-C3 alkylene, and R 42 is hydrogen, Even more preferably: R 41 is C2 alkylene, R 42 is acetyl or methyl, or R 41 is C2 alkylene, R 42 is hydrogen.

[0023] In yet another embodiment, the cation is: [ka] (wherein X is a nitrogen atom or a phosphorus atom, Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen and / or phosphorus atoms; R6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R7 is C1-C4 alkyl. is selected from.

[0024] In yet another embodiment, the cation is: [ka] (In the formula, X1 and X2 may be the same or different and are nitrogen atoms or phosphorus atoms, Ring A is a five- or six-membered heterocycle; R6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R7 is C1-C4 alkyl. is selected from.

[0025] In formulae (I-2) and (I-2'), ring A is preferably a five- or six-membered heteroaromatic ring, more preferably a five-membered heteroaromatic ring, and even more preferably an imidazolium ring.

[0026] In formulae (I-2) and (I-2′), R6 is preferably: -R 61 OR 62 (In the formula, R 61 is C1-C4 alkylene, R 62 is hydrogen, C1-C2 alkyl or C1-C4 acyl; More preferably: -R 61 OR 62 (In the formula, R 61 is C1-C2 alkylene, R 62 is C1-C2 alkyl or C1-C2 acyl, or R 61 is C1-C4 alkylene, R 62 is hydrogen).

[0027] In formulae (I-2) and (I-2'), R7 is preferably a C1 to C2 alkyl, more preferably methyl.

[0028] In this specification, the term "hydrocarbon group" includes not only groups consisting of carbon and hydrogen atoms, but also groups which contain, in addition to carbon and hydrogen atoms, functional groups such as hydroxy groups, ether bonds, aldehyde groups, and carboxy groups.

[0029] In another preferred embodiment, the cation is: [ka] is selected from.

[0030] In the liquid composition according to the present invention, preferred specific combinations of cations and anions are, for example: -cholinium cation (Ia) and dihydrogen phosphate anion (IIa), -cholinium cation (Ia) and dihydrogen citrate anion (IIb), -cholinium cation (Ia) and thiocyanate anion (IIc), -cholinium cation (Ia) and bitartrate ion (IId), -cholinium cation (Ia) and bromide ion, -Acetylcholine cation (Ib) and chloride ion, -1-(2-hydroxyethyl)-3-methylimidazolium cation (Ic) and dihydrogen phosphate anion (IIa), -1-(2-Hydroxyethyl)-3-methylimidazolium cation (Ic) and chloride ion, and -N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium cation (Id) and bromide ion, Such specific combinations of cations and anions are particularly suitable for stabilizing membrane proteins, but are not limited to these.

[0031] The liquid composition according to the present invention preferably contains cations and anions in a molar ratio of 1: 1. By containing cations and anions in such a molar ratio, it is possible to provide a field more suitable for the analysis of membrane proteins.

[0032] The liquid composition according to the present invention contains an ion pair of anion and cation in, for example, a buffer solution. For example, a Tris-HCl buffer solution is used as the buffer solution. The concentration of the ion pair in the liquid composition is, for example, less than 50 mM.

[0033] In one embodiment, the membrane protein is an α-helical transmembrane protein. The present inventors have found that the hydrated ionic liquid containing the above-mentioned combination of the specific cation and the specific anion acts to stabilize the structure of a transmembrane protein in which the proportion of α-helix is ​​high. Unlike β-barrel transmembrane proteins, which have a similar stability to water-soluble proteins, α-helical transmembrane proteins have been considered difficult to fold in vitro. It was surprising that they were stabilized by a hydrated ionic liquid containing a combination of a specific cation and a specific anion. For example, it is believed that surfactants used in the past to dissolve membrane proteins achieve solubilization of membrane proteins by adjusting hydrophilic and hydrophobic sites, but the hydrated ionic liquid used in the present invention does not have a clear hydrophobic site, and therefore the solubilization mechanism is thought to be different from that of surfactants.

[0034] The α-helical transmembrane protein has, for example, 1 to 17, preferably 4 to 12, and more preferably 7 to 10 transmembrane domains in a monomer.

[0035] In the present specification, "stabilized" and "stable" in relation to a protein refer to the retention of the higher-order structure of a membrane protein, preferably the retention of the three-dimensional structure of a membrane protein. Specifically, "stabilized" and "stable" refer to the maintenance of the higher-order structure of a protein observed in the natural state, such as α-helix and β-sheet, and the higher-order structure and / or three-dimensional structure of a protein in the natural state are retained even in an in vitro environment in which the protein is extracted and purified by a method suitable for the protein, and refer to a structure formed in water, a saline solution of an appropriate concentration range, and various buffer solutions that satisfy the pH conditions commonly used in the field. In the case of a membrane protein, for example, the higher-order structure and / or three-dimensional structure formed in the presence of a surfactant of an appropriate type and concentration in water, a saline solution, a buffer solution, etc. The higher-order structure and / or three-dimensional structure of a protein can be measured by a method known in the art, for example, circular dichroism (CD) spectroscopy, ultraviolet-visible (UV)-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy, attenuated total reflection (ATR), differential scanning calorimetry (DSC), nuclear magnetic resonance (NMR), X-ray structural analysis, electron microscopy, high-speed atomic force microscopy, and fluorescence spectroscopy, but are not limited to these methods. The higher-order structure and / or three-dimensional structure of a protein may be measured by using these methods alone or in combination.

[0036] For example, peaks characteristic of an α-helix structure in a CD spectrum include a peak near 205-210 nm and a peak near 220-225 nm. The maintenance of the higher-order structure and / or three-dimensional structure of an α-helix can be confirmed by, for example, maintaining the peak intensity and / or shape as a result of correcting for concentration. The maintenance of the higher-order structure and / or three-dimensional structure can be confirmed by, for example, maintaining a shape having peaks near 208 nm and near 222 nm, which are characteristic of an α-helix structure. The maintenance of the peak intensity can be confirmed by, for example, maintaining the peak intensity at a certain wavelength (e.g., 208 nm) within the above wavelength range of the CD spectrum, which is, for example, 50% or more, 60% or more, 70% or more, or 75% or more of the peak intensity in the native state, preferably 80% or more, 85% or more, 90% or more, or 95% or more, more preferably equivalent to the peak intensity in the native state.

[0037] In the present specification, the stability of a membrane protein preferably includes thermal stability. Here, thermal stability is also referred to as heat resistance. In the present specification, with respect to a membrane protein, "stabilization" and "being stable" preferably refer to the membrane protein being dissolved while maintaining its higher-order structure, and the membrane protein having improved thermal stability. More preferably, "stabilization" and "being stable" refer to the membrane protein being dissolved while maintaining its three-dimensional structure, and the membrane protein having improved thermal stability. Thermal stability can be evaluated by performing measurements at different temperatures using any of the above-mentioned methods. Thermal stability can be confirmed, for example, by measuring the denaturation temperature of the protein. For example, when the denaturation temperature of the protein in the liquid composition according to the present invention is increased compared to the denaturation temperature of the protein in a buffer solution, the improved thermal stability is confirmed. When the membrane protein has a chromophore, the change in color may be confirmed visually or by a conventional device before and after heating.

[0038] In the present specification, the stability of a membrane protein preferably includes stability over time. Here, stability over time is also referred to as storage stability. In the present specification, with respect to a membrane protein, "stabilization" and "being stable" preferably refer to the membrane protein being dissolved while maintaining its higher-order structure, and the membrane protein being stable over time. More preferably, "stabilization" and "being stable" refer to the membrane protein being dissolved while maintaining its three-dimensional structure, and the membrane protein being stable over time. The stability over time can be evaluated by performing measurements at different times using the above-mentioned methods. For example, the temperature can be kept constant, and a certain time point is compared with a certain time after the time point, and the higher-order structure and / or three-dimensional structure can be confirmed to be maintained.

[0039] As used herein, with respect to a membrane protein, "stabilized" and "stable" more preferably refer to the membrane protein being dissolved while retaining its higher-order structure and / or three-dimensional structure, and also to the protein function being retained. The retention of the membrane protein function can be confirmed using a method appropriate for the type of protein. For example, the retention of the function of bacteriorhodopsin can be confirmed by measuring the photoreaction cycle.

[0040] In an even more preferred embodiment, "stabilized" and "stable" refer to the fact that the higher-order and / or three-dimensional structure of the membrane protein is maintained, the thermal stability of the membrane protein is improved, and the function of the membrane protein is maintained. In an even more preferred embodiment, "stabilized" and "stable" refer to the fact that the higher-order and / or three-dimensional structure of the membrane protein is maintained, the membrane protein has stability over time, and the function of the membrane protein is maintained.

[0041] In a more particularly preferred embodiment, the term "stabilization" and "being stable" of a membrane protein may also include the absence of change in higher-order structure and / or three-dimensional structure in response to an external stimulus, such as light irradiation, specifically laser irradiation, etc.

[0042] Another aspect of the present invention relates to a method for stabilizing a membrane protein, the stabilization method comprising adding the membrane protein to a liquid composition comprising the above-mentioned cation and the above-mentioned anion, the composition of which is the same as that described above for the liquid composition.

[0043] The stabilization method of the present invention may include stirring the liquid composition containing the membrane protein and / or adjusting the liquid temperature.

[0044] As described above, according to the present invention, a simple method for stabilizing membrane proteins, which have been difficult to obtain sufficient stability in a solution, is provided. Generally, when membrane proteins are isolated from biological membranes, they do not maintain their higher-order structure without the presence of an appropriate surfactant or lipid. It has also been found that isolated membrane proteins do not maintain their structure even in high concentrations of more common ionic liquids. However, it has been surprisingly discovered that by using the liquid composition according to the present invention, it is possible to dissolve membrane proteins in liquid while maintaining their higher-order structure.

[0045] The hydrated ionic liquid used in the present invention can be prepared simply by adding water molecules to an ionic liquid containing a specific cation and a specific anion, and the isolated membrane protein can be stably dissolved in the liquid simply by mixing the isolated membrane protein with this hydrated ionic liquid. In other words, the advantages of the present invention are that the field for dissolving the membrane protein can be easily prepared in a short time, the number of coexisting water molecules can be adjusted, and the membrane protein can be dissolved while maintaining its higher-order structure by mixing a membrane protein solution with the prepared hydrated ionic liquid.

[0046] In addition, the stability of a membrane protein can be improved simply by dissolving the membrane protein in a hydrated ionic liquid containing a specific ion pair and water, thereby reducing the burden of complicated work, skilled techniques, time required for preparation, etc. For example, according to the present invention, there is no need to control the orientation as in the case of using liposomes, and no operation after reconstruction as in the case of reconstruction using nanodisks is required.

[0047] Furthermore, since hydrated ionic liquids are hardly volatile even in an open environment, they can be used on a small scale for a long period of time. Therefore, it is expected that the membrane protein stabilized by the present invention can be developed as a binding compound screening technique that can be used for long-term studies. In other words, it is expected that the stabilized membrane protein can be used to efficiently screen compounds that interact with the membrane protein, for example, at room temperature in an open environment, and to analyze the binding strength of the compound. EXAMPLES

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0049] In the examples, the membrane proteins used were Haemophilus influenzae TehA (hereinafter referred to as "TehA"), which contains 10 transmembrane α-helices, and bacteriorhodopsin (hereinafter referred to as "bR"), which has a structure in which α-helices penetrate the membrane seven times and exchanges energy as a light-driven proton pump. bR is a pigment protein that contains the chromophore retinal. The two membrane proteins were prepared according to a previous report (Reference 3).

[0050] Example 1. TehA In the TehA experiment, an ion pair ([ch][dhp]) consisting of cholinium cation (cholinium, [ch]) and dihydrogenphosphate anion (dihydrogenphosphate, [dhp]) was used as the ionic liquid. Water was added so that there were 4 or 7 water molecules per [ch][dhp] ion pair to create hydrated [ch][dhp]. The buffer solution used was 150 mM NaCl, 20 mM Tris-HCl (pH 7.2-7.5), and 0.05% (w / v) DDM-anagrade. In addition, an aqueous solution containing [ch][dhp] at a concentration of 50 mM was also used for the thermal denaturation temperature measurement. The membrane protein was dissolved in each solvent to a concentration of approximately 500 μg / mL.

[0051] The prepared solution was incubated at 25, 40, 55, 60, 65, 70 and 85°C for 10 minutes, and then CD measurements were performed in the wavelength range of 200-250 nm (Figures 1, 2A and 2B). In the thermal denaturation temperature measurements in hydrated ionic liquids with different water contents, the change in CD intensity at 208 nm was tracked while changing the measurement temperature at a rate of 1°C / min. The temperature at which the spectral intensity was reduced to half was calculated as the denaturation temperature (Figures 3A and 3B).

[0052] As a result, it was confirmed that the spectrum in the hydrated ionic liquid was similar to that in the buffer solution, and it was revealed that the α-helical structure was maintained. Furthermore, the thermal denaturation temperature was higher in the hydrated ionic liquid than in the buffer solution, confirming the improvement of thermal stability. In particular, in the hydrated ionic liquid prepared so that there were four water molecules per ion pair, the temperature was about 30°C higher than in the buffer solution, confirming that dissolution in the hydrated [ch][dhp] greatly improved the thermal stability (Figure 3B).

[0053] Next, the ionic liquid and the crowding agent D-(+)-Trehalose Anhydrous shown below were hydrated. The hydrated mixture prepared is as follows. The numbers at the end of each formula indicate the number of water molecules per ion pair. ·[ch][dhp]-4, D-(+)-Trehalose Anhydrous (Tre-30), and ·Buffer solution TehA was added to each solvent to a concentration of 15 μM to prepare samples.

[0054] The secondary structure of TehA was confirmed by measuring circular dichroism (CD) for the prepared samples, and it was confirmed that TehA maintained a similar structure in [ch][dhp]-4 and Tre-30 as in the buffer solution. Furthermore, the change in CD intensity at 208 nm with temperature was tracked for these samples (Fig. 3C). The denaturation temperature was determined as the midpoint of the CD intensity change. The denaturation temperature of TehA dissolved in the buffer solution was 64°C, whereas the thermal denaturation temperature of TehA dissolved in [ch][dhp]-4 and Tre-30 was higher than that in the buffer solution. The thermal denaturation temperatures were 91°C for [ch][dhp]-4 and 73°C for Tre-30. An increase of approximately 30°C was confirmed for [ch][dhp]-4.

[0055] Next, hydrated [ch][dhp] was prepared so that the number of water molecules per ion pair was 1:4, 1:7, 1:10, 1:13, 1:17, and 1:20, and TehA was dissolved in each solution to perform CD measurements. The intensity of the CD spectrum at 208 nm was tracked while changing the temperature, and the midpoint of the intensity change was used as the thermal denaturation temperature for comparison (Fig. 3D and Fig. 3E). The thermal denaturation temperature in hydrated [ch][dhp], which was prepared with the fewest number of water molecules per ion pair (four molecules), was approximately 30°C higher than in the buffer solution, and the thermal denaturation temperature decreased as the number of water molecules increased.

[0056] Example 2.bR In the bR experiment, cholinium cation ([ch]) or acetylcholine cation ([Ach]) was used as the cation, and dihydrogen phosphate anion ([dhp]), dihydrogen citrate anion (dihydrogen citrate, [dhC]), thiocyanate ion ([SCN]), bromide ion (Br -, [Br]) or chloride ion (Cl - Specifically, the following ion pairs were used in the bR experiment. The numbers at the end of each pair indicate the number of water molecules per ion pair. - Ion pair of cholinium cation and dihydrogen phosphate anion ([CH][DHP]-3), - Ion pair of cholinium cation and dihydrogen citrate anion ([ch][dhC]-5), · Ion pair of cholinium cation and bromide ion ([ch][Br]-3) -Acetylcholine cation and chloride ion pair ([Ach][Cl]-3), - Ion pair of cholinium cation and thiocyanate ion ([ch][SCN]-3), and -Ion pair of tetrabutylammonium cation and dihydrogen phosphate anion ([N4444][dhp]-5),

[0057] In addition, a solution was prepared using polyethylene glycol 400 (PEG400), which is used as a molecular crowding agent.

[0058] The above solutions were prepared so that there were three water molecules per one ion pair, and for [ch][dhC] and [N4444][dhp], the ratio was five water molecules per one ion pair from the viewpoint of solubility. The buffer solution used was 20 mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl and 0.25% (w / v) sodium deoxycholate.

[0059] The dissolution state of bR was visually observed and measured by UV-Vis (Fig. 4A, 4B, 4C, 5A, 5B). In addition, visual observation and spectroscopic measurements were performed while incubating in buffer and [ch][dhp] at 90°C (Fig. 6).

[0060] The time it took for the spectrum to halve was calculated to compare heat resistance. The half-life at 90°C was 5.3 minutes in the buffer solution, but 21.3 minutes in [ch][dhp], confirming approximately four times the stabilization (Figure 5B). It was also confirmed by visual inspection that the sample in the buffer solution became transparent after 4 minutes at 90°C, but the color was maintained in [ch][dhp] (Figure 6).

[0061] In a photoabsorption experiment using flash excitation (Reference 6), the bR photoreaction cycle was analyzed by observing the absorbance changes at 410, 510, 570, and 630 nm after irradiation with a 532 nm nanosecond pulse laser at 2.0 mJ / pulse at 25°C (dots in Fig. 7A and Fig. 7B). Each absorbance change could be reproduced by the sum of six exponential functions (solid lines in Fig. 7A and Fig. 7B), and the time constants (seconds) were as shown in Fig. 8. After the measurements, the UV-visible absorption spectra before and after laser irradiation were compared (Fig. 9).

[0062] As a result of the above test, the ratio of the time constants of the light absorption change in the buffer solution and the hydrated ionic liquid (HIL / B column in Figure 8) was found to be about 0.5 to 0.6 for the first half of time constants 1 to 3, which are not related to the movement of hydrogen ions, and was about twice as fast in the hydrated ionic liquid. For the second half of time constants 4 to 6, which are related to the movement of hydrogen ions, the ratio was about 4.8 to 17, and was about 5 to 17 times slower in the hydrated ionic liquid. This is thought to be due to the low hydrogen ion concentration in the hydrated ionic liquid. Despite these differences, it was confirmed that the ionic liquid showed a photoreaction cycle similar to that in the buffer solution. In addition, in the hydrated ionic liquid, almost no fading was observed due to the approximately 360 laser irradiations used in the measurement, indicating that it is stable against light irradiation (Figure 9).

[0063] In addition, hydrated ionic liquids were prepared using various cations and anions, as shown below, and it was confirmed that bR has the same structure as in the buffer solution. The numbers at the end of the numbers indicate the number of water molecules per ion pair. The ionic liquids were either synthesized or purchased. - Ion pair of cholinium cation and dihydrogen phosphate anion ([CH][DHP]-4), - Ion pair of cholinium cation and bitartrate ion ([ch][bit]-6), - Ion pair of cholinium cation and bromide ion ([ch][Br]-3), -Acetylcholine cation and chloride ion pair ([Ach][Cl]-3), ·N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium cation and bromide ion pair ([DEME][Br]-3), 1-(2-Hydroxyethyl)-3-methylimidazolium cation and chloride ion pair ([C2mimOH][Cl]-4), -(2-Hydroxyethyl)-3-methylimidazolium cation and dihydrogen phosphate anion ([C2mimOH][dhp]-3), ·Betain-7

[0064] In addition, polyethylene glycol 400 (PEG400), which is used as a molecular crowding agent, was used to prepare three water molecules per ion pair (PEG400-3).

[0065] Samples were prepared in each solvent so that the concentration of TehA was 15 μM. No precipitation of bR was observed in any of the hydrated ionic liquids prepared. Visible-ultraviolet spectroscopy was performed on each solution.

[0066] In the case of bR in the buffer solution, a large absorption band was observed around 560 nm due to retinal, and a small, broad absorption band was observed around 400 nm (Fig. 10A). In [ch][dhp]-4, a spectrum similar to that in the buffer solution was observed (Fig. 10B), and in Betain-7, a change in structure was suggested (Fig. 10C). The peak near 400 nm, which is characteristic of the spectrum suggesting a change, closely resembles the spectrum of free retinal, suggesting the isolation of retinal from bR.

[0067] Next, the spectral changes accompanying temperature changes in the hydrated ionic liquid were tracked, and the thermal denaturation temperatures of each were calculated. bR dissolved in the hydrated ionic liquid was incubated in an incubator from 25°C to 100°C, with the temperature increasing by 5°C per 5 minutes, and the spectrum was measured every 5 minutes using the UV-vis method to observe the absorbance at the maximum absorption wavelength. The thermal denaturation temperatures were calculated as the ratio of the absorbance at each measurement temperature to the absorbance at 25°C, and plotted. The temperature at which the absorbance reached 50% was taken as the thermal denaturation temperature (Figure 11). It was confirmed that the thermal denaturation temperature was higher in all hydrated ionic liquids than in the buffer solution.

[0068] Further investigation was carried out into the decay tendency of absorbance observed in [ch][dhp]-4, [ch][bit]-6, and [C2mimOH][dhp]-3, which showed a significant increase in thermal denaturation temperature compared to that in buffer (Fig. 12A-D). In [C2mimOH][dhp]-3, the decay rate observed with increasing temperature was slow, but the decay started at a relatively low temperature of around 45°C (Fig. 12C). In contrast, the decay start temperature of [ch][dhp]-4 was around 55°C (Fig. 12A). [ch][dhp]-4 had a higher decay start temperature, at which structural changes began to occur, and was more stable (Fig. 12D).

[0069] Furthermore, to examine the water content of hydrated [ch][dhp], solutions were prepared with 4, 7, and 15 molecules per ion pair, and the thermal denaturation temperature of each was measured by UV-vis spectroscopy (Figures 13A-13C). The thermal denaturation temperature at which the absorbance before incubation in hydrated [ch][dhp] decayed to 50% was approximately 80°C for 1:4, approximately 83°C for 1:7, and approximately 68°C for 1:15, and it was confirmed that there was little difference between 1:4 and 1:7, but was significantly lower for 1:15. It was suggested that the stability improved as the amount of free water in hydrated [ch][dhp] decreased.

[0070] In the prior art, for example, the effects of ionic liquids and osmolytes on the structure and oxidative stress of the membrane protein bacteriorhodopsin have been investigated through experiments and simulations (Reference 4). The ions used here are completely different from those used in the present invention in terms of structure and concentration. Although no significant effect on the structure of bR has been observed, no improvement in stability has been confirmed.

[0071] There have also been cases where the heat resistance of membrane proteins has been successfully improved by using calculations to design heat resistance and then introducing mutations (Reference 5). A heat-resistant mutant of the membrane protein bR was designed by selecting the site to introduce the mutation in the extramembrane region, calculating the free energy of the denatured state, predicting structural changes due to the mutation, and determining candidates for heat-resistant mutations. However, the success rate of the design was only 40%, and some mutants failed to be made heat-resistant.

[0072] According to the present invention, the stability of membrane proteins is improved by dissolving the membrane proteins in a hydrated ionic liquid containing a specific ion pair and water, and therefore the burden of complicated work, skilled techniques, and the time required for preparation can be reduced. In addition, since the membrane proteins are dissolved in a liquid, all protein molecules can be uniformly stabilized. Furthermore, since the proteins can be dissolved and stabilized in a liquid, long-term use for screening binding compounds is expected.

[0073] References TIFF0007615425000016.tif94161

Claims

1. (a) Below: 【Chemistry 1】 (wherein X is a nitrogen atom or a phosphorus atom; R 1 , R 2 and R 3 are hydrogen or C1-C4 alkyl; R 41 is C1-C4 alkylene, and R 42 is hydrogen, C1-C2 alkyl, or C1-C4 acyl. or the following: 【Chemistry 2】 (Wherein, X is a nitrogen atom, Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen atoms; R 6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R 7 is C1-C4 alkyl. and a cation selected from (b) the following: 【Chemistry 3】 An anion selected from and 3 to 13 water molecules are included for each pair of the cation and the anion; A liquid composition for stabilizing membrane proteins that is a hydrated ionic liquid.

2. The cation is selected from the group consisting of: 【Chemistry 4】 (In the formula, X １ and X ２ is a nitrogen atom, Ring A is a five- or six-membered heterocycle; R ６ is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain, R ７ is C1-C4 alkyl. The liquid composition of claim 1 , wherein the liquid composition is selected from:

3. The cation is selected from the group consisting of: 【Chemistry 5】 The liquid composition of claim 1 , wherein the liquid composition is selected from:

4. The liquid composition according to claim 1 or 2, further comprising a membrane protein.

5. The liquid composition according to claim 1 or 2, wherein the membrane protein is an alpha-helical transmembrane protein.

6. The liquid composition of claim 5, wherein the alpha-helical transmembrane protein has 1 to 17 transmembrane domains.

7. 1. A method for stabilizing a membrane protein comprising adding the membrane protein to a liquid composition comprising a cation and an anion, (a) the cation is: 【Chemistry 6】 (wherein X is a nitrogen atom or a phosphorus atom; R 1 , R 2 and R 3 are hydrogen or C1-C4 alkyl; R 41 is C1-C4 alkylene, and R 42 is hydrogen, C1-C2 alkyl, or C1-C4 acyl. or the following: 【Chemistry 7】 (Wherein, X is a nitrogen atom, Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen atoms; R 6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R 7 is C1-C4 alkyl. is selected from (b) the anion is 【Chemistry 8】 is selected from the liquid composition contains 3 to 13 water molecules per pair of the cation and the anion; The method, wherein the liquid composition is a hydrated ionic liquid.

8. The method of claim 1, wherein the cation is: 【Chemistry 9】 (wherein X 1 and X 2 are nitrogen atoms; Ring A is a five- or six-membered heterocycle; R 6 is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain; R 7 is C1-C4 alkyl. The method of claim 7, wherein the compound is selected from the group consisting of 9. The method of claim 8, wherein the cation is: 【Chemistry 10】 The method of claim 7, wherein the compound is selected from the group consisting of

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