JP7615425B2 - Liquid compositions and methods for stabilizing membrane proteins - Patents.com - Google Patents

Description

The present invention relates to a liquid composition and method for stabilizing membrane proteins.

Membrane proteins are mainly found on cell membranes and play important biological roles. They are considered to be particularly important targets in drug discovery research, but they are difficult to handle outside of the body, limiting the scope of research. Specifically, membrane proteins are expressed in low amounts, and unlike water-soluble proteins, they have low solubility in solution, making it difficult to achieve sufficient stability in solution.

The use of liposomes and lipid nanodiscs has been proposed to stabilize membrane proteins isolated using surfactants, etc. (Non-Patent Documents 1 and 2).

However, the production of liposomes requires procedures 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 for preparation. Depending on the size of the vesicles, physicochemical measurements of liposomes can also be difficult. Furthermore, multi-step procedures are required to evaluate the efficiency of membrane protein incorporation into liposomes. In addition, the orientation of membrane proteins in liposomes is not always uniform, and is not easy to control.

Lipid nanodiscs are disk-shaped particles composed of phospholipids and proteins modified from human-derived lipoprotein A. The preparation of lipid nanodiscs requires many days for the 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 the empty nanodiscs and/or excess membrane proteins.

As such, there is room for improvement in the technology for stably maintaining membrane proteins in vitro, and there is a need for a simple method for stabilizing membrane proteins that can replace conventional methods.

Methods Enzymol, 464, 211, 2009 Nature commun, 12, 2202, 2021

The present invention aims to provide a liquid composition and method for stabilizing membrane proteins.

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 liquids was examined by observing changes in the amide bands from infrared absorption spectra.

Next, the inventors investigated the application of hydrated ionic liquids to membrane proteins, which have structures and properties significantly different from water-soluble proteins, and surprisingly found that it is possible to dissolve membrane proteins in hydrated ionic liquids while maintaining their higher-order structure. Specifically, as a result of extensive 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 structure, 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.

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４は、水素またはＣ１～Ｃ４炭化水素基である（但し、Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４のすべてがアルキルであるカチオンを除く）。）
から選択されるカチオンと、
（ｂ）下記：

から選択されるアニオンと
を含み、
前記カチオンおよび前記アニオンの対１つに対して水分子が３～１３個含まれる、液体組成物；
［２］式（Ｉ）中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２およびＲ_３は、水素またはＣ１～Ｃ４アルキルであるか、または、Ｒ_１、Ｒ_２およびＲ_３が一緒に五員または六員環を形成しており、当該環は、Ｃ１～Ｃ４アルキルで置換されていてもよく、
Ｒ_４は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基である、［１］に記載の液体組成物；
［３］前記カチオンが、以下：

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２およびＲ_３は、水素またはＣ１～Ｃ４アルキルであり、
Ｒ_４１は、Ｃ１～Ｃ４アルキレンであり、Ｒ_４２は、水素、Ｃ１～Ｃ２アルキルもしくはＣ１～Ｃ４アシルである。）
から選択されるか、または、以下：

（式中、Ｘは、窒素原子またはリン原子であり、
環Ａは、五員または六員の複素環であり、さらに１つ以上の窒素原子および／またはリン原子を含んでいてもよく、
Ｒ_６は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基であり、
Ｒ_７は、Ｃ１～Ｃ４アルキルである。）
から選択される、［１］または［２］に記載の液体組成物；
［４］前記カチオンが、下記：

（式中、Ｘ_１およびＸ_２は同じであっても異なっていてもよく、窒素原子またはリン原子であり、
環Ａは、五員または六員の複素環であり、
Ｒ_６は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基であり、
Ｒ_７は、Ｃ１～Ｃ４アルキルである。）
から選択される、［３］に記載の液体組成物。
［５］前記カチオンが、下記：

から選択される、［１］～［４］のいずれか一項に記載の液体組成物；
［６］膜タンパク質を更に含む、［１］～［５］のいずれか一項に記載の液体組成物；
［７］前記膜タンパク質が、αヘリックス膜貫通型タンパク質である、［６］に記載の液体組成物；
［８］前記αヘリックス膜貫通型タンパク質が、１～１７個の膜貫通ドメインを有する、［７］に記載の液体組成物；
［９］カチオンとアニオンとを含む液体組成物に、膜タンパク質を添加することを含む、膜タンパク質を安定化する方法であって、
（ａ）前記カチオンが、下記：

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４は、水素またはＣ１～Ｃ４炭化水素基である（但し、Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４のすべてがアルキルであるカチオンを除く）。）
から選択され、
（ｂ）前記アニオンが、下記：

から選択され、
前記液体組成物中に、前記カチオンおよび前記アニオンの対１つ当たりに対して水分子が３～１３個含まれる、方法。 That is, the present invention is as follows:
[1] (a) the following:

(wherein X is a nitrogen atom or a phosphorus atom;
R ₁ , R ₂ , R ₃ and R ₄ are hydrogen or a C1-C4 hydrocarbon group (excluding cations in which R ₁ , R ₂ , R ₃ and R ₄ are all alkyl).
and a cation selected from
(b) the following:

and an anion selected from
a liquid composition comprising 3 to 13 water molecules for each pair of said cation and said anion;
[2] In formula (I), X is a nitrogen atom or a phosphorus atom,
R ₁ , R ₂ and R ₃ are hydrogen or C1-C4 alkyl, or R ₁ , R ₂ and R ₃ together form a 5- or 6-membered ring, which may be substituted with C1-C4 alkyl;
The liquid composition according to [1], wherein R ₄ is a C1-C4 hydrocarbon group having at least one oxygen atom in the chain;
[3] The cation is any one of the following:

(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:

(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],
[4] The cation is selected from the group consisting of:

(wherein X ₁ and X ₂ may be the same or different and are a nitrogen atom or a phosphorus atom;
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:

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

(wherein X is a nitrogen atom or a phosphorus atom;
R ₁ , R _{2 ,} R ₃ and R ₄ are hydrogen or a C1-C4 hydrocarbon group (excluding cations in which R ₁ , R ₂ , R ₃ and R ₄ are all alkyl).
is selected from
(b) the anion is

is selected from
The method of claim 1, wherein the liquid composition comprises 3 to 13 water molecules for each pair of the cation and the anion.

The present invention provides a liquid composition and method for stabilizing membrane proteins.

FIG. 1 shows the CD spectra of TehA in buffer and in hydrated ionic liquid. FIG. 1 shows the CD spectrum change with temperature of TehA in a buffer solution. FIG. 1 shows the CD spectrum change with temperature of TehA in a hydrated ionic liquid. FIG. 1 shows CD spectra of TehA in a buffer solution and in hydrated ionic liquids with different numbers of water molecules. 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. FIG. 1 shows the change in CD spectral intensity (208 nm) of TehA in buffer, [ch][dhp]-4, and Tre-30 with temperature. 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. 1 shows the denaturation temperatures of TehA in a buffer solution and in [ch][dhp] hydrated ionic liquids with different numbers of water molecules. FIG. 1 shows bR dissolved in buffer and in hydrated ionic liquid. FIG. 1 shows bR dissolved in buffer, PEG and various hydrated ionic liquids. FIG. 1 shows bR dissolved in a buffer solution and in various hydrated ionic liquids. FIG. 1 shows the UV-visible absorption of bR in buffer and in hydrated ionic liquid. FIG. 1 shows the time course of bR incubated at 90° C. in a buffer solution and in a hydrated ionic liquid. FIG. 1 shows the color change of bR in buffer and in hydrated ionic liquid upon heating. FIG. 1 shows the time course of optical absorption by bR in a hydrated ionic liquid upon flash excitation. FIG. 1 shows the time course of light absorption upon flash excitation of bR in a buffer solution. FIG. 1 shows an outline of the photoreaction cycle of bR and the corresponding time constants. FIG. 1 shows the time constants in the photocycle of bR in buffer and hydrated ionic liquid. FIG. 1 shows the change in UV-visible absorption of bR in hydrated ionic liquid before and after flash measurement. FIG. 1 shows the UV-visible absorption of bR in buffer. FIG. 1 shows the UV-visible absorption of bR in [ch][dhp]-4. FIG. 1 shows the UV-visible absorption of bR in Betain-7. FIG. 1 shows the thermal denaturation temperatures of bR in buffer solutions and each hydrated ionic liquid. FIG. 13 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-4. FIG. 13 shows the temperature change in UV-visible absorption of bR in [ch][bit]-6. FIG. 1 shows the UV-visible absorption of bR in [C2mimOH][dhp]-3. FIG. 1 shows the thermal denaturation temperatures of 50% and 80% bR in each hydrated ionic liquid. FIG. 13 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-4. FIG. 13 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-7. FIG. 13 shows the temperature dependence of UV-visible absorption of bR in [ch][dhp]-15.

The following is a detailed description of the embodiments for carrying out 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 gist of the present invention.

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, "ionic liquid (IL)" is non-volatile and flame-retardant, and has the characteristic of being liquid over a wide temperature range, so 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 was necessary to modify the biomolecule surface with a polymer or the like, or to use an additive. On the other hand, "hydrated ionic liquid (Hy IL)" is a mixture of 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 secondary structures such as α-helices and β-strands, and structures of higher dimensions than these structures, and may include tertiary structures and/or quaternary structures in addition to secondary structures. 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.

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４は、水素またはＣ１～Ｃ４炭化水素基である（但し、Ｒ_１、Ｒ_２、Ｒ_３およびＲ_４のすべてがアルキルであるカチオンを除く）。）
から選択されるカチオンと、
（ｂ）下記：

から選択されるアニオンと
を含む、液体組成物に関する。 The first aspect of the present invention comprises:
(a) the following:

(wherein X is a nitrogen atom or a phosphorus atom;
R ₁ , R ₂ , R ₃ and R ₄ are hydrogen or a C1-C4 hydrocarbon group (excluding cations in which R ₁ , R ₂ , R ₃ and R ₄ are all alkyl).
and a cation selected from
(b) the following:

and an anion selected from the group consisting of

The liquid composition of 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. Furthermore, 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, 4 to 13 water molecules are contained per ion pair. In another embodiment, 3 to 10 water molecules are contained per ion pair. In another embodiment, 3 to 7 water molecules are contained per ion pair.

The liquid composition of the present invention can stabilize membrane proteins by containing a small number of water molecules, specific cations, and specific anions. Therefore, the liquid composition of the present invention may further contain a membrane protein. The liquid composition of the present invention uses organic salts as cations and/or anions, so that the interaction between ions is small and the liquid composition has a structure that can be designed, making it easier to control the solvent properties than conventional solvents such as water and organic solvents.

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２およびＲ_３は、水素またはＣ１～Ｃ４アルキルであるか、または、Ｒ_１、Ｒ_２およびＲ_３が一緒に五員または六員環を形成しており、当該環は、Ｃ１～Ｃ４アルキルで置換されていてもよく、
Ｒ_４は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基である。）
から選択される。 In one embodiment, the cation is:

(wherein X is a nitrogen atom or a phosphorus atom;
R ₁ , R ₂ and R ₃ are hydrogen or C1-C4 alkyl, or R ₁ , R ₂ and R ₃ together form a 5- or 6-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.

（式中、Ｘは、窒素原子またはリン原子であり、
Ｒ_１、Ｒ_２およびＲ_３は、水素またはＣ１～Ｃ４アルキルであり、
Ｒ_４１は、Ｃ１～Ｃ４アルキレンであり、Ｒ_４２は、水素、Ｃ１～Ｃ２アルキルもしくはＣ１～Ｃ４アシルである。）
から選択される。 In another embodiment, the cation is:

(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.
is selected from.

In formula (I-1), R ₁ , R ₂ and R ₃ are preferably hydrogen or C1 to C2 alkyl, more preferably C1 to C2 alkyl.

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

（式中、Ｘは、窒素原子またはリン原子であり、
環Ａは、五員または六員の複素環であり、さらに１つ以上の窒素原子および／またはリン原子を含んでいてもよく、
Ｒ_６は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基であり、
Ｒ_７は、Ｃ１～Ｃ４アルキルである。）
から選択される。 In yet another embodiment, the cation is:

(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.

（式中、Ｘ_１およびＸ_２は同じであっても異なっていてもよく、窒素原子またはリン原子であり、
環Ａは、五員または六員の複素環であり、
Ｒ_６は、鎖内に少なくとも１つの酸素原子を有するＣ１～Ｃ４炭化水素基であり、
Ｒ_７は、Ｃ１～Ｃ４アルキルである。）
から選択される。 In yet another embodiment, the cation is:

(wherein X ₁ and X ₂ may be the same or different and are a nitrogen atom or a phosphorus atom;
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.

In formulas (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.

In formulae (I-2) and (I-2′), R ₆ is preferably:
-R ₆₁ OR ₆₂ (wherein R ₆₁ is C1-C4 alkylene and R ₆₂ is hydrogen, C1-C2 alkyl or C1-C4 acyl);
More preferably:
-R ₆₁ OR ₆₂ , where R ₆₁ is a C1-C2 alkylene and R ₆₂ is a C1-C2 alkyl or C1-C2 acyl, or R ₆₁ is a C1-C4 alkylene and R ₆₂ is hydrogen.

In formulae (I-2) and (I-2'), R ₇ is preferably a C1-C2 alkyl, more preferably methyl.

In this specification, the term "hydrocarbon group" includes not only groups consisting of only carbon and hydrogen atoms, but also groups that contain functional groups in addition to carbon and hydrogen atoms, such as hydroxyl groups, ether bonds, aldehyde groups, and carboxyl groups.

から選択される。 In another preferred embodiment, the cation is:

is selected from.

In the liquid composition according to the present invention, specific preferred combinations of cations and anions include, 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.

The liquid composition of 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 suitable environment for the analysis of membrane proteins.

The liquid composition of the present invention contains an ion pair of an anion and a cation, for example in 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.

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 a specific cation and a specific anion acts to stabilize the structure of a transmembrane protein in which α-helices account for a large proportion. 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, surfactants that have been used to dissolve membrane proteins are thought to 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 is therefore thought to have a different solubilization mechanism from surfactants.

An 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.

In this specification, "stabilized" and "stable" in relation to a protein refer to the retention of the higher-order structure of the membrane protein, preferably the retention of the three-dimensional structure of the membrane protein. Specifically, "stabilized" and "stable" refer to the maintenance of the higher-order structure of the protein observed in the natural state, such as α-helix and β-sheet, and the higher-order structure and/or three-dimensional structure of the protein in the natural state are retained even in an in vitro environment after extraction and purification by a method suitable for each protein, and refer to structures formed in water, saline of an appropriate concentration range, and various buffer solutions that satisfy pH conditions commonly used in the field. In the case of membrane proteins, 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, saline, 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 using these techniques alone or in combination.

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, for example, by the maintenance of peak intensity and/or shape as a result of concentration correction. The maintenance of the higher-order structure and/or three-dimensional structure can be confirmed, for example, by the maintenance of a shape having peaks near 208 nm and near 222 nm, which are characteristic of an α-helix structure ... peak intensity at a wavelength (e.g., 208 nm) within the above wavelength range of the CD spectrum being, 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.

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 mean that the membrane protein is dissolved while maintaining its higher-order structure, and that the thermal stability of the membrane protein is improved. More preferably, "stabilization" and "being stable" mean that the membrane protein is dissolved while maintaining its three-dimensional structure, and that the thermal stability of the membrane protein is improved. Thermal stability can be evaluated by performing measurements at different temperatures using any of the methods exemplified above. Thermal stability can be confirmed, for example, by measuring the denaturation temperature of the protein. For example, if the denaturation temperature of the protein in the liquid composition of the present invention is increased compared to the denaturation temperature of the protein in a buffer solution, the thermal stability is confirmed to be improved. If the membrane protein has a chromophore, the change in color before and after heating may be confirmed visually or by a conventional device.

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 mean that the membrane protein is dissolved while maintaining its higher-order structure, and that the membrane protein has stability over time. More preferably, "stabilization" and "being stable" mean that the membrane protein is dissolved while maintaining its three-dimensional structure, and that the membrane protein has stability over time. The stability over time can be evaluated by performing measurements at different points in time using the above-mentioned method. For example, the temperature can be kept constant, and a comparison can be made between a certain point in time and a certain time after the passage of time from that point in time to confirm that the higher-order structure and/or three-dimensional structure is maintained.

As used herein, with respect to membrane proteins, "stabilized" and "stable" more preferably mean that the membrane protein is dissolved while retaining its higher-order structure and/or three-dimensional structure, and that the function of the protein is maintained. The retention of the function of the membrane protein 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.

In an even more preferred embodiment, "stabilized" and "stable" refer to the fact that the higher-order structure and/or the 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 structure and/or the 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.

In a more particularly preferred embodiment, "stabilization" and "being stable" of a membrane protein may also include not causing changes in higher-order structure and/or three-dimensional structure in response to an external stimulus. An example of an external stimulus is light irradiation, specifically laser irradiation, etc.

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 aforementioned cation and the aforementioned anion. The composition of the liquid composition is as described above for the liquid composition.

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

As described above, the present invention provides a simple method for stabilizing membrane proteins, which have traditionally been difficult to achieve sufficient stability in solution. In general, 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 of the present invention, it is possible to dissolve membrane proteins in liquid while maintaining their higher-order structure.

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 by mixing a membrane protein solution with the prepared hydrated ionic liquid, the protein can be dissolved while maintaining its higher-order structure.

In addition, the stability of membrane proteins can be improved simply by dissolving them in a hydrated ionic liquid containing a specific ion pair and water, which can reduce the burden of cumbersome work, skilled techniques, and the time required for preparation. For example, according to the present invention, there is no need to control the orientation as in the case of using liposomes, and no post-reconstruction operations are required as in the case of reconstruction using nanodisks.

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

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

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).

Example 1. TehA
In the TehA experiment, an ion pair ([ch][dhp]) of cholinium cation ([ch]) and dihydrogenphosphate anion ([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]. 150 mM NaCl, 20 mM Tris-HCl (pH 7.2-7.5), and 0.05% (w/v) DDM-anagrade were used as the buffer. In addition, an aqueous solution in which the concentration of [ch][dhp] was adjusted to 50 mM was also used for the thermal denaturation temperature measurement. The membrane protein was dissolved in each solvent so that the concentration was about 500 μg/mL.

The prepared solutions were incubated at temperatures of 25, 40, 55, 60, 65, 70 and 85°C for 10 minutes, after which CD measurements were performed in the wavelength range of 200-250 nm (Fig. 1, Fig. 2A and Fig. 2B). In measurements of thermal denaturation temperatures 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 halved was calculated as the denaturation temperature (Fig. 3A and Fig. 3B).

As a result, a spectrum similar to that in the buffer solution was confirmed in the hydrated ionic liquid, demonstrating 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 improved thermal stability. In particular, in the hydrated ionic liquid prepared so that there were four water molecules per ion pair, the temperature was approximately 30°C higher than in the buffer solution, confirming that dissolution in the hydrated [ch][dhp] significantly improved thermal stability (Figure 3B).

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 number indicate the number of water molecules per ion pair.
・[ch][dhp]-4,
TehA was added to each of the solvents of D-(+)-Trehalose Anhydrous (Tre-30) and the buffer solution to a concentration of 15 μM, and samples were prepared.

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 change was tracked for these samples (Figure 3C). The midpoint obtained by the change in CD intensity was taken as the denaturation temperature, and 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 confirmed to be higher than that in the buffer solution. The thermal denaturation temperature was 91°C for [ch][dhp]-4 and 73°C for Tre-30. An increase of approximately 30°C was confirmed for [ch][dhp]-4.

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 to perform CD measurements. The intensity of the CD spectrum at 208 nm obtained while changing the temperature was tracked, and the midpoint of the intensity change was used as the thermal denaturation temperature for comparison (Figures 3D and 3E). The thermal denaturation temperature in hydrated [ch][dhp] adjusted with the fewest number of water molecules per ion pair (4 molecules) was approximately 30°C higher than in buffer solution, and the thermal denaturation temperature decreased as the number of water molecules increased.

Example 2. bR
In the bR experiments, 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 ^- , [Cl]) were used as the anions. Specifically, the following ion pairs were used in the bR experiments. 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)
- Ion pair of acetylcholine cation and chloride ion ([Ach][Cl]-3),
- Ion pair of cholinium cation and thiocyanate ion ([ch][SCN]-3),
and an ion pair of tetrabutylammonium cation and dihydrogen phosphate anion ([N4444][dhp]-5),

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

The above solutions were prepared so that there were three water molecules per ion pair, and for [ch], [dhC], and [N4444], [dhp], the ratio was five water molecules per 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.

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

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).

In an experiment on photoabsorption by 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 Figures 7A and 7B). Each absorbance change could be reproduced by the sum of six exponential functions (solid lines in Figures 7A and 7B), and the time constants (seconds) were as shown in Figure 8. After the measurements, the UV-visible absorption spectra before and after laser irradiation were compared (Figure 9).

As a result of the above test, the ratio of the time constants of the light absorption change in the buffer solution and in 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 concentration of hydrogen ions 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. Furthermore, in the hydrated ionic liquid, almost no fading was observed after approximately 360 laser irradiations used in the measurement, indicating that it is stable against light irradiation (Figure 9).

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 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),
- Ion pair of acetylcholine cation and chloride ion ([Ach][Cl]-3),
- Ion pair of N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium cation and bromine ion ([DEME][Br]-3),
- Ion pair of 1-(2-hydroxyethyl)-3-methylimidazolium cation and chloride ion ([C2mimOH][Cl]-4),
-(2-Hydroxyethyl)-3-methylimidazolium cation and dihydrogen phosphate anion ion pair ([C2mimOH][dhp]-3),
・Betain-7

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).

Samples were prepared so that the concentration of TehA in each solvent 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.

In the case of bR in buffer solution, a large absorption band was observed near 560 nm due to retinal, and a small, broad absorption band was observed near 400 nm (Figure 10A). In [ch][dhp]-4, a spectrum similar to that in buffer solution was observed (Figure 10B), and in the case of Betaine-7, a change in structure was suggested (Figure 10C). The peak just before 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.

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.

Further investigation was carried out into the observed decay tendency of absorbance in [ch][dhp]-4, [ch][bit]-6, and [C2mimOH][dhp]-3, which were confirmed to have significantly higher thermal denaturation temperatures than in buffer (Fig. 12A-D). In [C2mimOH][dhp]-3, the decay rate observed with increasing temperature was slow, but decay began at a relatively low temperature of around 45°C (Fig. 12C). In contrast, the decay onset temperature for [ch][dhp]-4 was around 55°C (Fig. 12A). [ch][dhp]-4 had a higher and more stable decay onset temperature at which structural changes began to occur (Fig. 12D).

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] decays to 50% is approximately 80°C for 1:4, approximately 83°C for 1:7, and approximately 68°C for 1:15, indicating that there is little difference between 1:4 and 1:7, but is significantly lower at 1:15. This suggests that the less free water there is in hydrated [ch][dhp], the greater the stability.

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 both structure and concentration. Although no significant effect on the structure of bR has been observed, no improvement in stability has been confirmed.

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 domain, 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.

According to the present invention, the stability of membrane proteins is improved by dissolving them in a hydrated ionic liquid containing a specific ion pair and water, which reduces the burden of cumbersome work, skilled techniques, and the time required for preparation. In addition, since the membrane proteins are dissolved in liquid, all protein molecules can be uniformly stabilized. Furthermore, since the proteins can be dissolved and stabilized in liquid, they are expected to be used for the long term for screening binding compounds.

References

Claims (9)

(a) The following:

(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:

(Wherein, X is a nitrogen atom,
Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen atoms;
R6 is _a C1-C4 hydrocarbon group having at least one oxygen atom in the chain;
R7 is C1-C4 alkyl _.
and a cation selected from
(b) the following:

and an anion selected from
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 . The cation is selected from the group consisting of:

(wherein _X1 and _X2 are nitrogen 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 of claim 1 , wherein the liquid composition is selected from: The cation is selected from the group consisting of:

The liquid composition of claim 1 , wherein the liquid composition is selected from: The liquid composition according to claim 1 or 2, further comprising a membrane protein. The liquid composition according to claim 1 or 2 , wherein the membrane protein is an alpha-helical transmembrane protein. The liquid composition of claim 5 , wherein the alpha-helical transmembrane protein has 1 to 17 transmembrane domains. 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 :

(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:

(Wherein, X is a nitrogen atom,
Ring A is a five- or six-membered heterocycle, which may further contain one or more nitrogen 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
(b) the anion is

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 . The cation is selected from the group consisting of:

(Wherein, 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 method of claim 7, wherein the compound is selected from the group consisting of The cation is selected from the group consisting of:

The method of claim 7, wherein the compound is selected from the group consisting of