Fluorescent probe for GABA detection and method for manufacturing same

Abstract

In the present invention, a fluorescence-labeled Pf622 protein includes a fluorescent dye-bound amino acid residue to which a fluorescent dye is bound, and is configured such that: (1) when GABA is not bound to Pf622 protein, or when GABA is bound to Pf622 protein, another amino acid residue is present within 2 nm of the fluorescent dye-bound amino acid residue; and (2) when GABA is bound to Pf622 protein, if compared to the case where GABA is not bound to Pf622 protein, the distance between the fluorescent dye-bound amino acid residue and the other amino acid residue becomes 0.1 nm or less, or becomes 0.1 nm or more.

Description

Fluorescent probe for detecting GABA and method for producing the same

[0001] The present disclosure relates to a fluorescent probe for detecting GABA and a method for manufacturing the same. According to the present disclosure, it is possible to provide a fluorescent probe for detecting GABA that has an excellent rate of change in fluorescence intensity.

[0002] GABA is the main inhibitory neurotransmitter in the brain and is essential for maintaining higher brain functions. Abnormalities in GABA transmission in the brain have been observed in various neuropsychiatric disorders and aging. Therefore, accurate measurement of GABA content in biological tissues such as the brain would be useful for elucidating the molecular pathological mechanisms of neuropsychiatric disorders. Furthermore, it is believed that the development of new drugs and new treatment methods using GABA as an indicator would become possible.

[0003] Conventionally, known methods for measuring GABA include a method using a FRET probe (Non-Patent Document 1), a method using a hybrid probe (Non-Patent Document 2), and a method using a fluorescent protein probe (Patent Document 1). Also proposed is a selective labeling method in which a fluorescent dye is bound to an amino acid residue, such as a lysine residue, a methionine residue (Non-Patent Document 3), a tyrosine residue (Non-Patent Document 4), or a tryptophan residue (Non-Patent Document 5), in a protein used in a probe.

[0004] US Patent Application Publication No. 2022 / 0050103

[0005] Journal of the American Chemical Society, 2012 (USA), Vol. 134, p. 19026-19034 Nature Methods, 2019 (UK), Vol 16, p. 763-770 Nature, 2018 (UK), vol. 562, p. 563-568 Journal of the American Chemical Society, 2010 (USA), vol. 132, p. 1523-1525 Journal of the American Chemical Society, 2016 (USA), vol. 138, p. 10798-10801

[0006] However, the fluorescent probes that have been used to measure GABA do not exhibit a sufficient rate of change in fluorescence intensity, and some of them exhibit fluctuations in fluorescence intensity due to the influence of pH.

[0007] Therefore, an object of the present disclosure is to provide a fluorescent probe for detecting GABA that has an excellent rate of change in fluorescence intensity. More specifically, an object of the present disclosure is to provide a fluorescent probe for detecting GABA that has an excellent rate of change in fluorescence intensity and exhibits stable fluorescence intensity that is not affected by pH.

[0008] As a result of extensive research, the present inventors have surprisingly found that by introducing a fluorescent dye into specific amino acid residues of the Pf622 protein, a fluorescent probe for detecting GABA can be obtained that exhibits an excellent rate of change in fluorescence intensity. Furthermore, the present inventors have found that the fluorescence intensity of the fluorescent probe for detecting GABA does not change with changes in pH, and that the probe has excellent stability against changes in pH value. The technology of the present disclosure has been developed based on these findings.

[0009] Accordingly, the present disclosure provides the following: [1] a fluorescently labeled Pf622 protein comprising a fluorescent dye-binding amino acid residue to which a fluorescent dye is bound, wherein (1) when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein, another amino acid residue is present within 2 nm of the fluorescent dye-binding amino acid residue, and (2) when GABA is bound to the Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and the other amino acid residue is 0.1 nm or more closer or 0.1 nm or more farther than when GABA is not bound to the Pf622 protein; [2] the fluorescently labeled Pf622 protein according to [1], wherein the fluorescent dye-binding amino acid residue is cysteine; [3] the fluorescently labeled Pf622 protein according to [1] or [2], which is a fluorescent probe for detecting GABA; [4] a method for producing a fluorescent probe for detecting GABA, comprising: (A1) a step of analyzing any two amino acid residues of the Pf622 protein by protein three-dimensional structure analysis, and selecting amino acid residues that are located within a distance of 2 nm when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein; (A2) a step of selecting amino acid residues that are 0.1 nm or more closer to or 0.1 nm or more further apart when the Pf622 protein and GABA are bound, compared to when GABA is not bound to the Pf622 protein; and (A3) a step of binding a fluorescent dye to the selected amino acid residues; [5] the production method according to [4], wherein the GABA-binding protein is Pf622 protein; [6] a method for measuring the concentration of GABA in a sample, comprising: (B1) a step of measuring the fluorescence intensity of the fluorescently labeled Pf622 protein according to any one of [1] to [4]; (B2) a step of contacting the sample with the fluorescently labeled Pf622 protein; (B3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (B2); and (B4) calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (B1) and the fluorescence intensity measured in the step (B3);[7] A method for screening a substance that affects the dynamics of GABA, comprising: (C1) adding the fluorescently labeled Pf622 protein according to any one of [1] to [4] to nerve cells or nerve tissue, or a culture thereof, and measuring the fluorescence intensity; (C2) contacting the nerve cells, the nerve tissue, or the culture with a candidate substance; (C3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (C2); and (C4) calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (C1) and the fluorescence intensity measured in the step (C3); [8] A method for screening a stimulus that affects the dynamics of GABA, comprising: (D1) adding the fluorescently labeled Pf622 protein to nerve cells or nerve tissue, or a culture thereof, and measuring the fluorescence intensity; (D2) applying a stimulus to the nerve cells, the nerve tissue, or the culture; (D3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (D2); (D4) a step of calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (D1) and the fluorescence intensity measured in the step (D3); [9] the method according to [7] or [8], wherein the GABA dynamics is GABA release or GABA removal;

[10] a kit comprising the fluorescently labeled Pf622 protein according to any one of [1] to [4] and a package insert containing instructions for use;

[11] the kit according to

[10] , for detecting GABA or for screening a substance or stimulus that affects GABA dynamics;

[0010] The technology disclosed herein can provide a fluorescent probe for detecting GABA that has an excellent rate of change in fluorescence intensity. Specifically, the technology disclosed herein can provide a fluorescent probe for detecting GABA that has an excellent rate of change in fluorescence intensity and exhibits stable fluorescence intensity that is not affected by pH.

[0011]

[0023] Figure 1 is a schematic diagram of a Pf622 protein containing a fluorescent dye-binding amino acid residue to which a fluorescent dye is bound. Figure 2 shows the free form of Pf622 protein to which GABA is not bound, and the bound form to which GABA is bound. Figure 3 shows the change in the rate of change in fluorescence intensity with changes in GABA concentration for fluorescently labeled Pf622 protein in which Janelia Fluor585 fluorescent dye (JF585) is bound to G183C. Figure 4 shows the change in fluorescence intensity with changes in pH for fluorescently labeled Pf622 protein in which fluorescent dye JF585 is bound to G183C.

[0012] <1> Fluorescent Probe for GABA Detection A first aspect of the present disclosure relates to a fluorescent probe for GABA detection. The fluorescent probe for GABA detection is a fluorescently labeled Pf622 protein containing a fluorescent dye-binding amino acid residue to which a fluorescent dye is bound ( FIG. 1 ). The Pf622 protein is a protein that binds to gamma-aminobutyric acid (hereinafter also referred to as "GABA"). The fluorescent dye-bound Pf622 protein exhibits a change in fluorescence intensity before and after binding to GABA, and thus GABA can be detected based on the change in fluorescence intensity.

[0013] In the fluorescently labeled Pf622 protein, (1) when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein, other amino acid residues are present within 2 nm of the fluorescent dye-binding amino acid residue, and (2) when GABA is bound to the Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues is 0.1 nm or more closer or 0.1 nm or more farther than when GABA is not bound to the Pf622 protein.

[0014] In the present disclosure, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein before and after GABA binding is, but not limited to, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, % or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600% or more, 610% or more, 620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more and 800% or more.

[0015] More specifically, when the concentration of GABA is 1 mM or more, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein before and after GABA binding is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 60 0% or more, 610% or more, 620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more and 800% or more.Furthermore, when the concentration of GABA is 10 mM or more, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein before and after GABA binding is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600% or more, 610% or more, 620% or more, 630% 0% or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600 % or more, 610% or more, 620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more, and 800% or more. Furthermore, when the GABA concentration is 10 to 20 mM, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein before and after GABA binding may also show the above-mentioned values.

[0016] In the present disclosure, the fluorescently labeled Pf622 protein may be used in vivo or in vitro, i.e., the fluorescently labeled Pf622 protein may be used both inside and outside an animal.

[0017] <<Pf622 Protein>> The Pf622 protein is a protein consisting of a 315 amino acid sequence represented by SEQ ID NO: 1, and as described above, binds to GABA. The Pf622 protein was identified as a homologue of the GABA-binding protein Atu4243 (Non-Patent Document 2).

[0018] γ-Aminobutyric Acid γ-Aminobutyric acid (GABA) is one of three structural isomers of aminobutyric acid: α-, β-, and γ-, and has been reported to function primarily as an inhibitory neurotransmitter. In the central nervous system of vertebrates, it is mainly present in the cerebral neocortex, hippocampus, cerebellum, spinal cord, etc. It also acts as a neurotransmitter in arthropods and crustaceans. At the synapse, GABA is released from the presynaptic membrane and exerts its action by binding to GABA receptor proteins on the postsynaptic membrane. GABA is produced in the brain by the removal of the α-carboxyl group of glutamic acid through a reaction with glutamic acid decarboxylase. Three subtypes of GABA receptors that bind to γ-aminobutyric acid are known, each of which is a GABA receptor. Ａ Receptor, GABA Ｂ Receptors and GABA Ｃ are called receptors.

[0019] Abnormalities in GABA transmission in the brain have been observed in various neuropsychiatric disorders and aging. Therefore, if the GABA content in biological tissues such as the brain could be precisely measured, it is expected that understanding the molecular pathological mechanisms of neuropsychiatric disorders and developing new drugs and new treatment methods using GABA as an indicator will become possible.

[0020] <<Fluorescent Dye>> The fluorescent dye is not particularly limited, but examples thereof include Janelia Fluor (JF) fluorescent dye, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, tetramethylenerhodamine isothiocyanate (TRITC), sulforhodamine 101 acid chloride, fluorescamine, Cy3, Cy5, Cy5.5, Cy7, TAMRA, SPY, SiR, MaP, Alexa, BODIPY, DyLight, ATTO, MegaStokes, Dapoxyl, Dancyl chloride, rhodol, and Nile Red. Furthermore, examples of Janelia Fluor (JF) fluorescent dyes include JF585, JF646, JF549, JF503, JF519, JF525, JF526, JF608, JF635, and JF669.

[0021] (Binding of Amino Acid Residues to Fluorescent Dyes) In the present disclosure, binding of amino acid residues in the fluorescently labeled Pf622 protein to fluorescent dyes can be performed using methods commonly used in the field, but is not particularly limited thereto. Examples of amino acid residues to which fluorescent dyes can be bound include selective labeling methods for lysine residues, methionine residues (Non-Patent Document 3), tyrosine residues (Non-Patent Document 4), and tryptophan residues (Non-Patent Document 5).

[0022] Alternatively, the amino acid residues of the wild-type (hereinafter also referred to as "unmodified") Pf622 protein can be substituted with cysteine, and the cysteine's SH group (sulfhydryl group) can be used to bind to a fluorescent dye. For example, a maleimide group can be introduced into the fluorescent dye, and the amino acid residue can be bound to the fluorescent dye via the bond between the sulfhydryl group and the maleimide group.

[0023] The substitution of an amino acid residue with cysteine ​​can be performed using amino acid substitution methods commonly used in the field, such as PCR, genome editing methods such as CRISPR / Cas, and site-specific mutagenesis of a base sequence by gene synthesis.

[0024] A linker can also be used to bond the amino acid residue to the fluorescent dye. The linker is not particularly limited, but examples include linkers containing a hydrocarbon group having 1 to 8 carbon atoms (C1, C2, C3, C4, C5, C6, C7, or C8) and linkers having a basic structure such as phenol or PEG.

[0025] As described above, the amino acid residue and the fluorescent dye can be bonded to the SH group (sulfhydryl group) of cysteine ​​using a maleimide group or the like bonded to the fluorescent dye. In this case, the fluorescent dye phore and the maleimide group can be bonded by a conventional method. Specifically, the maleimide group and the fluorescent dye phore can be bonded using a group commonly used in this field, such as an amide bond (-NH-CO-) or an ester bond (-O-CO-), for example, -CH ２ -CH ２Binding can be achieved by —NH—CO—, etc. These groups do not affect the effect of the Pf622 protein.

[0026] <<Distance between Fluorescent Dye-Binding Amino Acid Residue and Other Amino Acid Residues>> In the fluorescently labeled Pf622 protein, (1) when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein, other amino acid residues are present within 2 nm of the fluorescent dye-binding amino acid residue. Furthermore, in the fluorescently labeled Pf622 protein, (2) when GABA is bound to the Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues is 0.1 nm or more closer or 0.1 nm or more farther than when GABA is not bound to the Pf622 protein. The distance between the fluorescent dye-binding amino acid residue and other amino acid residues is specifically the linear distance between the α-carbon of the fluorescent dye-binding amino acid residue and the α-carbon of the other amino acid residue. Hereinafter, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues may be simply referred to as the "distance between two amino acid residues."

[0027] The distance between the fluorescent dye-binding amino acid residue and other amino acid residues is described below with reference to FIG. 2 . The Pf622 protein undergoes a structural change upon GABA binding. The structure without GABA is called the "free form," and the structure with GABA bound is called the "bound form." In the free form and bound form shown in FIG. 2 , other amino acid residues are present within 2 nm of the fluorescent dye-binding amino acid residue. In the bound form, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues is either 0.1 nm or more closer or 0.1 nm or more farther. That is, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues changes by 0.1 nm or more between the free form and the bound form. Due to the change in the distance between the fluorescent dye-binding amino acid residue and other amino acid residues as described above, the fluorescently labeled Pf622 protein exhibits a significant change in fluorescence intensity.

[0028] Between the free form and the bound form of the fluorescently labeled Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and other amino acid residues may vary, for example, by 0.2 nm or more, 0.3 nm or more, 0.4 nm or more, 0.5 nm or more, 0.6 nm or more, and 0.7 nm or more.

[0029] In another aspect, in a fluorescently labeled Pf622 protein, (1) when GABA is not bound to the Pf622 protein and when GABA is bound to the Pf622 protein, other amino acid residues are present within 2 nm of the fluorescent dye-binding amino acid residue, and (2) when GABA is bound to the Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and the other amino acid residue is 0.1 nm or more closer or 0.1 nm or more farther than when GABA is not bound to the Pf622 protein.

[0030] (Determining Amino Acid Distances by Protein 3D Structure Analysis) The distance between the fluorescent dye-binding amino acid residue and other amino acid residues can be determined by protein 3D structure analysis, but is not limited to this, as follows. For example, using free-form and bound-form protein 3D structure data, the linear distance between the α-carbon of the fluorescent dye-binding amino acid residue and the α-carbon of other amino acid residues is measured. These linear distances between α-carbons can be measured using protein 3D structure display and analysis software such as PyMOL and UCSF Chimera, or can be determined from the coordinate information of the protein 3D structure data. Free-form and bound-form protein 3D structure data can be obtained from experiments such as X-ray crystallography and nuclear magnetic resonance (NMR), and may also be obtained from databases such as Protein Data Bank or protein 3D structure prediction software such as AlphaFold2 and Rosetta. For proteins of interest for which it is difficult to obtain protein 3D structure data for both the free and bound forms, protein 3D structure analysis of the free and bound forms of another protein with a similar 3D structure can be performed, and the linear distance between the α-carbon atoms of the free and bound forms of the protein of interest can be estimated based on the homologous amino acids in the primary amino acid sequence of the other protein with a similar 3D structure to the protein of interest. In the Examples described below, protein 3D structure data for the free and bound forms of Atu4243, a homolog of Pf622 with a similar protein 3D structure, was obtained from the Protein Data Bank (PDB ID: 4EQ7, 4EUO), and the linear distance between the α-carbon of the fluorescent dye-binding amino acid residue and the α-carbon of other amino acid residues was measured.

[0031] As a result of determining the amino acid distances by the protein three-dimensional structure analysis, the 279 amino acids shown in Table 1 below were selected as other amino acid residues that satisfy the above conditions (1) and (2).

[0032]

[0033] Fluorescently labeled Pf622 proteins in which a fluorescent dye is bound to any of the 279 amino acids exhibit an excellent rate of change in fluorescence intensity. In the present disclosure, fluorescently labeled Pf622 proteins may exclude those in which a fluorescent substance is labeled at the 278th amino acid.

[0034] In one embodiment, the fluorescently labeled Pf622 protein is selected from the group consisting of F6, V7, S8, W9, G10, G11, S12, T13, Q14, D15, A16, Q17, K18, Q19, A20, W21, A22, D23, P24, S26, K27, A28, S29, G30, Q35, D36, G37, P38, T39, D40, Y41, G42, K43, L44, K45, A46, V48, W55, D56, V57, V58, D59, V60, E61, A62, D63, F64, A65, L66, R67, A68, A69, A70, E71, G72, L73, L74, E 75, P76, L77, D78, F79, S80, V81, I82, Q83, R84, D85, K86, I87, D88, P89, R90 , F91, V92, S93, D94, H95, G96, V97, G98, S99, F100, L101, F102, S103, F104, V105, L106, G107, Y108, N109, E110, G111, K112, L113, G114, A115, S116, P1 18, Q119, L124T127, T129, Y130, P131, G132, K133, R134, A135, L136, Y137, K 138, W139, P140, S141, P142, G143, V144, L145, E146, L147, A148, L149, L15 0, P155, A156, D157, K158, L159, Y160, P161, L162, D163, L164, D165, R166, A167, F168, K169, K170, L171, D172, I174, K175, K176, D177, I178, V179, W1 80, W181, G182, G183, G184, A185, Q186, S187, Q188, Q189, L190, L191, A192, S193, G194, E195, V196, S197, M198, G199, Q200, F201, W202, N203, G204, R2 05, I206, H207, A208, L209, Q210, E211, D212, G213, A214, P215, V216, G217, V218, S219, W220, K221, Q222, N223, L224, V225, M226, A227, D228, I229, L2 30, V231, V232, P233, K234, T236, K239, A240, A242, M243, K244, F245, L246,A247, A249, A252, K253, G254, Q255, D256, D257, F258, S259, N260, L261, T262, A263, Y264, A265, P266, V26 7, N268, I269, D270, S271, V272, Q273, R274, L275, D276, S277, L279, A280, P281, N282, L283, P284, T285, A A fluorescent dye is bound to any one of 286, Y287, V288, K289, D290, Q291, I292, T293, L294, D295, F296, A297, Y298, W299, A300, K301, N302, G303, P304, A305, I306, A307, T308, R309, W310, N311, E312, W313, L314, V315 and K316.

[0035] In another embodiment, the fluorescently labeled Pf622 protein has a fluorescent dye attached to any one of A16, Q19, D36, K43, G183, Q188, A208, L209, D270, S271, V272, R274, and L275.

[0036] In another embodiment, the fluorescently labeled Pf622 protein has a fluorescent dye bound to any one of A16C, Q19C, D36C, G183C, A208, S271C, and L275C.

[0037] Without intending to be bound by any theory, the present inventors presume that the region of amino acids 260 to 290 of Pf622 is a region that undergoes a significant structural change before and after GABA binding, and consider this region to be an important labeling site for binding a fluorescent dye.

[0038] <<Rate of Change in Fluorescence Intensity>> In the present disclosure, the fluorescently labeled Pf622 protein exhibits an excellent rate of change in fluorescence intensity. Although not limited thereto, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, or 400% or more. , 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600% or more, 610% or more, 620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more and 800% or more.

[0039] More specifically, when the GABA concentration is 1 mM or more, the rate of change in fluorescence intensity of the fluorescently labeled Pf622 protein is, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 260% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600% or more, 610% or more, 620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more, and 800% or more, and when the GABA concentration is 10 mM or more, for example, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more, 210% or more, 220% or more, 230% or more, 240% or more, 250% or more, 26 0% or more, 270% or more, 280% or more, 290% or more, 300% or more, 310% or more, 320% or more, 330% or more, 340% or more, 350% or more, 360% or more, 370% or more, 380% or more, 390% or more, 400% or more, 410% or more, 420% or more, 430% or more, 440% or more, 450% or more, 460% or more, 470% or more, 480% or more, 490% or more, 500% or more, 510% or more, 520% ​​or more, 530% or more, 540% or more, 550% or more, 560% or more, 570% or more, 580% or more, 590% or more, 600% or more, 610% or more,620% or more, 630% or more, 640% or more, 650% or more, 660% or more, 670% or more, 680% or more, 690% or more, 700% or more, 710% or more, 720% or more, 730% or more, 740% or more, 750% or more, 760% or more, 770% or more, 780% or more, 790% or more, and 800% or more.

[0040] The rate of change in fluorescence intensity can be calculated by the following formula: Rate of change in fluorescence intensity (%)=100×{(fluorescence intensity after GABA binding)−(fluorescence intensity before GABA binding)} / (fluorescence intensity before GABA binding).

[0041] (Effect of pH on Fluorescence Intensity) In the present disclosure, the fluorescently labeled Pf622 protein exhibits stable fluorescence intensity that is not affected by pH. Therefore, the amount of GABA can be accurately measured even in an environment where the pH changes, such as the brain environment where the pH fluctuates. Furthermore, in drug efficacy evaluation and compound screening using the amount of GABA in a biological sample as an index, the amount of GABA can be accurately measured without being affected by pH fluctuations caused by the compound.

[0042] <<Action>> In the present disclosure, the mechanism by which the fluorescently labeled Pf622 protein exhibits an excellent change in fluorescence intensity is not intended to be bound by any theory, but can be assumed as follows. That is, the fluorescence intensity changes due to physicochemical interactions between the fluorescent dye and adjacent amino acids. It is assumed that this interaction changes significantly with a change in distance of less than a nanometer, and in particular, a change in distance of less than 0.1 nm results in a small change in fluorescence intensity, but a change in distance of 0.1 nm or more results in a large change in fluorescence intensity.

[0043] In another embodiment, the fluorescently labeled Pf622 protein exhibits a higher rate of change in fluorescence intensity and exhibits stable fluorescence intensity with reduced pH effects compared to a wild-type Pf622 protein, specifically a fluorescently labeled Pf622 protein including an unmodified Pf622 protein.

[0044] <2> Method for Manufacturing a Fluorescent Probe for GABA Detection A second aspect of the present disclosure relates to a method for manufacturing a fluorescent probe for GABA detection. The method for manufacturing a fluorescent probe for GABA detection includes the following steps: (A1) analyzing any two amino acid residues of a GABA-binding protein by protein 3D structure analysis and selecting amino acid residues that are located within 2 nm of each other when GABA is not bound to Pf622 protein or when GABA is bound to the GABA-binding protein; (A2) selecting amino acid residues that are 0.1 nm or more closer or 0.1 nm or more further apart when the GABA-binding protein and GABA are bound, compared to when GABA is not bound to the GABA-binding protein; and (A3) binding a fluorescent dye to the selected amino acid residues. The fluorescent probe for GABA detection obtained by the above method for manufacturing a fluorescent probe for GABA detection exhibits an excellent rate of change in fluorescence intensity. The GABA-binding protein is preferably Pf622 protein. Steps (A1) to (A3) will be explained in detail below for the case where the GABA-binding protein is Pf622 protein.

[0045] Step (A1): In step (A1), any two amino acid residues of the Pf622 protein are analyzed by protein 3D structure analysis, and amino acid residues that are within 2 nm of each other when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein are selected. The determination of the distance between two amino acid residues "when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein" by protein 3D structure analysis in step (A1) can be performed by the protein 3D structure analysis described in "<1> Fluorescent probe for GABA detection" above. That is, the distance between any two amino acid residues can be determined by protein 3D structure analysis.

[0046] Step (A2): In step (A2), amino acid residues are further selected from the two amino acid residues selected in step (A1) so that when Pf622 protein and GABA bind, the distance between the two amino acid residues is 0.1 nm or more closer or 0.1 nm or more further apart than when GABA is not bound to Pf622 protein. The change in the distance between the two amino acid residues in step (A2) between "when Pf622 protein and GABA bind" and "when GABA is not bound to Pf622 protein" can be determined by the protein three-dimensional structure analysis described in "<1> Fluorescent probe for GABA detection" above. The amino acid residues selected in step (A2) are those listed in Table 1.

[0047] <<Step (A3)>> In step (A3), a fluorescent dye is bound to the selected amino acid residue. The fluorescent dye may be any of those described in "<1> Fluorescent probe for GABA detection" above. Furthermore, the method for binding the fluorescent dye to the selected amino acid residue may be any of the methods described in "<1> Fluorescent probe for GABA detection" above, without any limitation.

[0048] <3> Method for measuring GABA concentration As described above, in the present disclosure, the fluorescently labeled Pf622 protein can be used as a fluorescent probe for detecting GABA. By using the fluorescently labeled Pf622 protein as a fluorescent probe for detecting GABA, the GABA concentration in a sample can be measured. Therefore, a further aspect of the present disclosure relates to a method for measuring the GABA concentration in a sample.

[0049] The method for measuring the GABA concentration in a sample includes the following steps: (B1) measuring the fluorescence intensity of a fluorescently labeled Pf622 protein; (B2) contacting the sample with the fluorescently labeled Pf622 protein; (B3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after step (B2); and (B4) calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in step (B1) and the fluorescence intensity measured in step (B3).

[0050] The above-mentioned measuring method can be carried out either in vivo or in vitro.The above-mentioned measuring method is preferably carried out in vitro.The above-mentioned measuring method can be used to measure the GABA concentration in, for example, blood and cerebrospinal fluid.Therefore, the sample can include, for example, blood and cerebrospinal fluid.The sample can also include, for example, cells and tissue sections.

[0051] In addition, fluorescently labeled Pf622 protein can be used as a fluorescent probe for detecting GABA to measure the dynamics of GABA in the brain and other biological tissues that secrete GABA.For example, high-resolution microscopes such as confocal microscopes and two-photon microscopes can be used to measure the dynamics of GABA in real time.

[0052] Furthermore, the fluorescently labeled Pf622 protein can be used as a fluorescent probe for detecting GABA to elucidate biological functions involving GABA. For example, the fluorescently labeled Pf622 protein can be used to elucidate the role of GABA in brain function, physiological function, etc. Specifically, by measuring GABA released by GABAergic neurons, glial cells, B cells, etc. in real time, it can be used to elucidate how GABA derived from each cell affects biological functions. It can also be used to elucidate the role of GABA in cognitive functions such as memory and learning, physiological functions such as sleep and wakefulness, and immune functions such as biodefense responses.

[0053] Furthermore, it can also be used to analyze the mechanisms of GABA release and elimination by observing the dynamics and / or measuring the concentration of GABA using in vivo imaging of GABA in animals lacking functional molecules involved in GABA release and elimination, or in the presence of inhibitors of these functional molecules.

[0054] Fluorescently labeled Pf622 protein can also be used to analyze the pathological mechanisms of psychiatric disorders and neurodegenerative diseases, as well as to develop diagnostic and therapeutic methods. GABA is also associated with diseases such as psychiatric disorders and neurodegenerative diseases, so investigating the dynamics and concentration changes of GABA in pathological conditions is expected to be useful for analyzing pathological mechanisms, such as how GABA is involved in the onset and progression of the disease, and for developing new diagnostic and therapeutic methods. For example, in vivo fluorescent imaging of GABA can be applied to analyze pathological mechanisms in the brain tissues of model animals with diseases such as schizophrenia, Parkinson's disease, Alzheimer's disease, autism, depression, and epilepsy.

[0055] Furthermore, fluorescently labeled Pf622 protein can be used to evaluate the effects and safety of drugs and external stimuli. By evaluating how candidate drugs and external stimuli affect GABA dynamics, the effects and safety of new pharmaceuticals and therapies targeting GABA can be evaluated. More specifically, fluorescently labeled Pf622 protein can be used to evaluate the effects and safety of cognitive enhancing drugs and antidepressant drugs, electrical and magnetic stimulation therapies such as transcranial direct current stimulation (tDCS) and transcranial magnetic stimulation (TMS), and other physical stimuli. Such evaluations can be performed not only in vivo but also outside the animal body, for example, in cultured cells and tissue slices.

[0056] <4> Screening Method for Substances Affecting GABA Dynamics Yet another aspect of the present disclosure relates to a method for screening for substances that affect GABA dynamics. The screening method for substances that affect GABA dynamics includes the following steps: (C1) adding fluorescently labeled Pf622 protein to neurons or neural tissue, or a culture thereof, and measuring the fluorescence intensity; (C2) contacting the neurons, neural tissue, or culture with a candidate substance; (C3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after step (C2); and (C4) calculating the rate of change in fluorescence intensity from the fluorescence intensity measured in step (C1) and the fluorescence intensity measured in step (C3). Here, the substance may be, for example, a compound. A candidate substance selected based on the obtained rate of change in fluorescence intensity can be used as a drug candidate. A substance can be selected as a drug candidate based on a reference value for the rate of change in fluorescence intensity, determined, for example, based on a negative control substance. The screening method may be performed either in vitro or in vivo, but is preferably performed in vitro. In the above screening method, the GABA dynamics is, for example, the release and removal of GABA, specifically, the release and removal of GABA from tissues and cells. When the GABA dynamics is the release of GABA, the candidate substance may be selected based on the items derived from the obtained rate of change in fluorescence intensity, such as the amount and frequency of GABA release.

[0057] <5> Screening Method for Stimuli Affecting GABA Dynamics Yet another aspect of the present disclosure relates to a method for screening stimuli affecting GABA dynamics. The screening method for stimuli affecting GABA dynamics includes the following steps: (D1) adding fluorescently labeled Pf622 protein to neurons or neural tissue, or a culture thereof, and measuring the fluorescence intensity; (D2) applying a stimulus to the neurons, neural tissue, or culture; (D3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after step (D2); and (D4) calculating the rate of change in fluorescence intensity from the fluorescence intensity measured in step (D1) and the fluorescence intensity measured in step (D3). Here, the stimulus affecting GABA dynamics refers to an external action that affects GABA dynamics. The stimulus may be, for example, a physical stimulus such as an electrical stimulus, a magnetic stimulus, a mechanical stimulus, a thermal stimulus, or a light stimulus. More specifically, examples of stimuli include, but are not limited to, transcranial magnetic stimulation (TMS), transcranial direct current stimulation (tDCS), and deep brain stimulation (DBS). Stimulation may be invasive or non-invasive, with non-invasive stimulation being preferred. Stimuli may be selected as candidates for therapy based on, for example, a reference value for the rate of change in fluorescence intensity determined based on a negative control. The screening method may be performed either in vitro or in vivo, with ex vivo being preferred. The same applies to GABA dynamics as described above in "<4> Screening method for substances affecting GABA dynamics."

[0058] <6> Kit Yet another aspect of the present disclosure relates to a kit. The kit includes a fluorescently labeled Pf622 protein and an attached document containing instructions for use. The kit is preferably a kit for detecting GABA or for screening for substances or stimuli that affect GABA dynamics.

[0059] As described above, the technology disclosed herein can provide a GABA-detecting fluorescent probe with an excellent rate of change in fluorescence intensity. The use of such a GABA-detecting fluorescent probe allows for the evaluation of the efficacy and safety of candidate drugs and physical stimulation therapies using GABA as an indicator, which is expected to be useful in the development of new therapeutic approaches. More specifically, the technology disclosed herein can provide a GABA-detecting fluorescent probe with an excellent rate of change in fluorescence intensity and stable fluorescence intensity that is not affected by pH. For example, in the brain, extracellular pH fluctuates with neural activity such as the release of neurotransmitters. By using a GABA-detecting fluorescent probe that exhibits stable fluorescence intensity that is not affected by pH, the molecular pathological mechanisms of GABA-related neuropsychiatric disorders can be analyzed even in the brain environment where pH fluctuates. Furthermore, because the GABA level in a biological sample can be used as an indicator for drug efficacy evaluation and compound screening without being affected by the pH-changing effects of compounds, the probe is expected to be useful in the development of new therapeutic agents in the field of drug discovery research.

[0060] The present disclosure will be specifically described below using examples, but these are not intended to limit the scope of the present disclosure in any way.

[0061] 1. Analysis Example: Protein Structure Analysis and Selection of Amino Acid Residues In this analysis example, steps (A1) and (A2) of the method for producing a fluorescent probe for GABA detection were performed. That is, protein structure analysis was performed for the cases where GABA was not bound to Pf622 protein and where GABA was bound to Pf622 protein, and amino acid residues that met the conditions were selected. The specific procedure is as follows.

[0062] Although only the free form of Pf622 protein 3D structure data is available from the Protein Data Bank (PDB ID: 6DGV), the bound form is unavailable due to lack of registration information. Therefore, we obtained the free and bound protein 3D structure data for Atu4243, a Pf622 homolog with a similar protein structure, from the Protein Data Bank (PDB ID: 4EQ7, 4EUO). We then measured the linear distance between the α-carbon of the fluorescent dye-binding amino acid residue and the α-carbon of other amino acid residues. Next, based on the homologous amino acids of Atu4243 and Pf622 in the primary amino acid sequence, we estimated the linear distance between the α-carbon of the free and bound forms of Atu4243 as the linear distance between the α-carbon of the free and bound forms of Pf622. From this estimate, 279 amino acid residues that met the criteria were selected.

[0063] 2. Preparation of Fluorescently Labeled Pf622 Protein Example 1 In this example, A16 of the 279 amino acid residues selected in the analysis example was substituted with cysteine ​​and labeled with the fluorescent dye JF585 according to the procedure described below. Specifically, a point mutation substituting A16 with cysteine ​​was first introduced into a plasmid containing the Pf622 gene by inverse PCR. Next, the plasmid in which A16 of the Pf622 protein was substituted with cysteine ​​was introduced into E. coli BL21(DE3) strain, and Pf622 protein containing the A16C mutation was produced in the E. coli. The E. coli was then lysed with a detergent, and the protein was purified using a histidine tag linked to the C-terminus of the Pf622 protein. JF585 fluorescent dye with a maleimide group attached was added to the purified Pf622 protein solution to obtain Pf622 protein labeled with JF585 at the A16 position. The fluorescent dye was bound to the SH group (sulfhydryl group) of cysteine ​​via a maleimide group bound to the fluorescent dye. The fluorescent dye and the maleimide group are represented by the formula: -CO-NH-CH ２ -CH ２ The bond was made via the - group.

[0064] Example 2 In this example, of the amino acid residues selected in the above analytical example, K18 was substituted with cysteine ​​and labeled with the fluorescent dye JF585. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was K18 instead of A16.

[0065] Example 3 In this example, of the amino acid residues selected in the above analytical example, Q19 was substituted with cysteine, and the Pf622 protein was labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was Q19 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0066] Example 4 In this example, of the amino acid residues selected in the above analytical example, D36 was substituted with cysteine ​​and labeled with the fluorescent dye JF585. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was D36 instead of A16.

[0067] Example 5 In this example, of the amino acid residues selected in the above analytical example, G182 was substituted with cysteine ​​and labeled with the fluorescent dye JF525. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was G182 instead of A16.

[0068] Example 6 In this example, of the amino acid residues selected in the above analytical example, G183 was substituted with cysteine, and the Pf622 protein was labeled with each of the fluorescent dyes JF525, JF549, JF585, and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was G183 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, or JF635.

[0069] Example 7 In this example, of the amino acid residues selected in the above analytical example, A185 was substituted with cysteine ​​and labeled with the fluorescent dye JF525. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was A185 instead of A16, and the fluorescent dye JF525 was used for labeling instead of the fluorescent dye JF585.

[0070] Example 8 In this example, of the amino acid residues selected in the above analytical example, A208 was substituted with cysteine, and the Pf622 protein was labeled with each of the fluorescent dyes JF525, JF549, JF585, and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was A208 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, or JF635.

[0071] Example 9 In this example, of the amino acid residues selected in the above analytical example, L209 was substituted with cysteine ​​and labeled with the fluorescent dyes JF549 and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was L209 instead of A16, and that labeling was performed with the fluorescent dye JF549 or JF635 instead of the fluorescent dye JF585.

[0072] Example 10 In this example, of the amino acid residues selected in the above analytical example, A214 was substituted with cysteine ​​and labeled with the fluorescent dye JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was A214 instead of A16, and the fluorescent dye JF635 was used for labeling instead of the fluorescent dye JF585.

[0073] Example 11 In this example, of the amino acid residues selected in the above analytical example, D270 was substituted with cysteine ​​and labeled with the fluorescent dyes JF585 and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was D270 instead of A16, and the protein was labeled with the fluorescent dye JF585 or JF635.

[0074] Example 12 In this example, of the amino acid residues selected in the above analytical example, S271 was substituted with cysteine ​​and labeled with the fluorescent dyes JF585 and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was S271 instead of A16, and the protein was labeled with the fluorescent dye JF585 or JF635.

[0075] Example 13 In this example, of the amino acid residues selected in the above analytical example, R274 was substituted with cysteine, and the Pf622 protein was labeled with the fluorescent dyes JF585 and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was R274 instead of A16, and the protein was labeled with the fluorescent dye JF585 or JF635.

[0076] Example 14 In this example, of the amino acid residues selected in the above analytical example, L275 was substituted with cysteine ​​and labeled with the fluorescent dyes JF585 and JF635. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was L275 instead of A16, and the protein was labeled with the fluorescent dye JF585 or JF635.

[0077] Comparative Example 1 In this example, of the amino acid residues not selected in the above analytical examples, M47 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was changed to M47 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0078] Comparative Example 2 In this example, of the amino acid residues not selected in the above analytical examples, A123C was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was changed from A16 to A123, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0079] Comparative Example 3 In this example, of the amino acid residues not selected in the above analytical examples, D126 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was changed from A16 to D126 and labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0080] Comparative Example 4 In this example, of the amino acid residues not selected in the above analytical examples, K128 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue in the Pf622 protein was changed to K128 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0081] Comparative Example 5 In this example, of the amino acid residues not selected in the above analytical examples, V154 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was V154 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0082] Comparative Example 6 In this example, of the amino acid residues not selected in the above analytical examples, T173 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was changed from A16 to T173 and labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0083] Comparative Example 7 In this example, of the amino acid residues not selected in the above analytical examples, S250 was substituted with cysteine ​​and labeled with each of the fluorescent dyes JF525, JF549, JF585, JF635, and JF646. A labeled Pf622 protein was obtained by the same procedure as in Example 1, except that the fluorescent dye-labeled amino acid residue of the Pf622 protein was S250 instead of A16, and the protein was labeled with the fluorescent dye JF525, JF549, JF585, JF635, or JF646.

[0084] 3. Measurement of Fluorescence Intensity of Fluorescently Labeled Pf622 Protein and Calculation of Rate of Change in Fluorescence Intensity The fluorescently labeled Pf622 proteins obtained in Examples 1 to 14 and Comparative Examples 1 to 7 were used as GABA detection probes, and their fluorescence intensities were measured according to the following procedure.

[0085] Fluorescently labeled Pf622 protein was added to a 96-well plate, and the fluorescence intensity before the addition of GABA was measured using a plate reader. Next, GABA was added to a final concentration of 1 mM or more, and the fluorescence intensity after the addition of GABA was measured using a plate reader. Finally, the rate of change in fluorescence intensity was calculated from the fluorescence intensities before and after the addition of GABA using the following formula: Rate of change in fluorescence intensity (%) = 100 × {(fluorescence intensity after the addition of GABA) - (fluorescence intensity before the addition of GABA)} ÷ (fluorescence intensity before the addition of GABA).

[0086] Table 2 shows the distance between two amino acid residues and the rate of change in fluorescence intensity for the fluorescently labeled Pf622 proteins of Examples 1 to 14 and Comparative Examples 1 to 7. In Table 2, "Dbound" and "Dfree" are the distance (nm) between the two amino acid residues before GABA binding and the distance (nm) between the two amino acid residues after GABA binding, respectively, and "ΔDmax" is the absolute value (nm) of the difference between Dbound and Dfree. In the Examples and Comparative Examples, the value of the rate of change in fluorescence intensity is the average of two or three experiments.

[0087] The fluorescently labeled Pf622 proteins of Examples 1 to 14 exhibited excellent rates of change in fluorescence intensity, whereas the fluorescently labeled Pf622 proteins of Comparative Examples 1 to 7 exhibited inferior rates of change in fluorescence intensity.

[0088]

[0089] 4. Study of the effect of GABA concentration on the rate of change in fluorescence intensity Using fluorescently labeled Pf622 protein (Example 6) in which the JF585 fluorescent dye was bound to G183C, the GABA concentration was varied from 0.1 to 10,000 μM, and the fluorescence intensity at each GABA concentration was measured using the method described above, and the rate of change in fluorescence intensity was calculated. Figure 3 shows the change in the rate of change in fluorescence intensity with changing GABA concentration.

[0090] 5. Study of the Effect of pH Changes on Fluorescence Intensity The effect of pH on the fluorescence intensity of fluorescently labeled Pf622 protein was studied using the procedure described below. Using fluorescently labeled Pf622 protein (Example 6) in which the fluorescent dye JF585 was bound to G183C, the fluorescence intensity was measured by changing the pH. Specifically, phosphate buffer solutions of pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.0, pH 7.5, and pH 8.0 were prepared. The fluorescently labeled Pf622 protein was added to each phosphate buffer solution at each pH, ​​and the fluorescence intensity of each solution was measured using a fluorescence plate reader. This fluorescence intensity value was designated the fluorescence intensity before GABA binding ("-GABA"). Next, GABA was added to a final concentration of 10 mM, and the fluorescence intensity of each solution was measured using a fluorescence plate reader. This fluorescence intensity value was designated the fluorescence intensity after GABA binding ("+GABA"). The fluorescence intensity at each pH is shown in Figure 4.

[0091] As shown in Figure 4, the fluorescently labeled Pf622 protein, in which the fluorescent dye JF585 was bound to G183C, maintained a nearly constant fluorescence intensity regardless of changes in pH. Specifically, in both the presence and absence of GABA, the fluorescence intensity hardly changed with changes in pH, and the rate of change in fluorescence intensity was stable. On the other hand, probes such as iGABASnFR, in which a fluorescent protein is bound to Pf622 protein as described in Supp. Fig. 14 of Non-Patent Document 2, emit almost no fluorescence at an acidic pH of around 5-6, but the fluorescence intensity fluctuates by several times or more at around pH 7-8.

[0092] The GABA detection fluorescent probe according to the technology of the present invention can accurately quantify minute amounts of GABA transiently released from synapses in the brain environment, where pH fluctuates with neural activity. Furthermore, in drug screening for neurological disorders, candidate compounds can induce significant changes in the activity of living cells, thereby fluctuating the pH of biological samples. Furthermore, the properties of the candidate compounds themselves can fluctuate the pH of biological samples. Therefore, the GABA detection fluorescent probe according to the technology of the present disclosure can be effectively used in compound screening or drug efficacy evaluation.

Claims

1. A fluorescently labeled Pf622 protein comprising a fluorescent dye-binding amino acid residue to which a fluorescent dye is bound, wherein (1) when GABA is not bound to the Pf622 protein or when GABA is bound to the Pf622 protein, another amino acid residue is present within 2 nm of the fluorescent dye-binding amino acid residue, and (2) when GABA is bound to the Pf622 protein, the distance between the fluorescent dye-binding amino acid residue and the other amino acid residue is 0.1 nm or more closer or 0.1 nm or more farther than when GABA is not bound to the Pf622 protein.

2. The fluorescently labeled Pf622 protein of claim 1, wherein the fluorescent dye-binding amino acid residue is cysteine.

3. The fluorescently labeled Pf622 protein according to claim 1 or 2, which is a fluorescent probe for detecting GABA.

4. A method for producing a fluorescent probe for detecting GABA, comprising: (A1) a step of analyzing any two amino acid residues of a GABA-binding protein by protein three-dimensional structure analysis, and selecting amino acid residues that are located within a distance of 2 nm when GABA is not bound to the GABA-binding protein or when GABA is bound to the GABA-binding protein; (A2) a step of selecting amino acid residues that are 0.1 nm or more closer to or 0.1 nm or more farther apart between the two amino acid residues when the GABA-binding protein and GABA are bound, compared to when GABA is not bound to the GABA-binding protein; and (A3) a step of binding a fluorescent dye to the selected amino acid residues.

5. The method of claim 4, wherein the GABA-binding protein is Pf622 protein.

6. A method for measuring a GABA concentration in a sample, comprising: (B1) a step of measuring the fluorescence intensity of the fluorescently labeled Pf622 protein described in claim 1 or 2; (B2) a step of contacting the sample with the fluorescently labeled Pf622 protein; (B3) a step of measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (B2); and (B4) a step of calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (B1) and the fluorescence intensity measured in the step (B3).

7. A method for screening a substance that affects GABA dynamics, comprising: (C1) adding the fluorescently labeled Pf622 protein described in claim 1 or 2 to nerve cells or nerve tissue or a culture thereof and measuring the fluorescence intensity; (C2) contacting the nerve cells or nerve tissue or the culture with a candidate substance; (C3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (C2); and (C4) calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (C1) and the fluorescence intensity measured in the step (C3).

8. A method for screening stimuli that affect GABA dynamics, comprising: (D1) adding fluorescently labeled Pf622 protein to nerve cells or nerve tissue, or a culture thereof, and measuring the fluorescence intensity; (D2) applying a stimulus to the nerve cells or nerve tissue, or the culture; (D3) measuring the fluorescence intensity of the fluorescently labeled Pf622 protein after the step (D2); and (D4) calculating a rate of change in fluorescence intensity from the fluorescence intensity measured in the step (D1) and the fluorescence intensity measured in the step (D3).

9. The method of claim 7, wherein the GABA dynamics is GABA release or GABA removal.

10. A kit comprising the fluorescently labeled Pf622 protein according to claim 1 or 2 and a package insert containing instructions for use.

11. A kit according to claim 10 for detecting GABA or for screening substances or stimuli that affect the dynamics of GABA.

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