Method and device for identifying structural polymorphism of fibrous protein or fibrous peptide

Abstract

To provide a method capable of easily and quickly identifying a structural polymorphism of a fibrous protein or a fibrous peptide.SOLUTION: A method for identifying a structural polymorphism of a fibrous protein or a fibrous peptide is provided, the method including the steps of: obtaining a fluorescence decay curve for a sample containing a fibrous protein or a fibrous peptide and thioflavin T; performing exponential fitting of four or more components based on a specific function G(t) on a function F(t) of the fluorescence decay curve to obtain fluorescence lifetime values τ1 to τn and weighting factors A1 to An (n is a natural number of 4 or more) of the respective exponential components; and identifying the structural polymorphism of the fibrous protein or the fibrous peptide on the basis of at least one or more values of the fluorescence lifetime values τ1 to τn and at least one or more values of the weighting factors A1 to An (a fluorescence lifetime value and a weighting factor in an exponential component derived from autofluorescence of thioflavin T are excluded).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for identifying structural polymorphisms of fibrous proteins or fibrous peptides. [Background technology]

[0002] Amyloids are abnormal fibrous aggregates formed due to changes in the three-dimensional structure or properties of peptides or proteins. Furthermore, fibrous proteins such as amyloids exhibit diverse fibrous structures (structural polymorphisms) even when the proteins that form them are the same. It is known that when proteins in the body form amyloids and accumulate in organs, various diseases are caused, and it is also known that the diseases caused by structural polymorphisms of amyloids vary.

[0003] For example, Non-Patent Documents 1 to 3 describe that the structure of tau amyloid differs between patients with Alzheimer's disease, Pick's disease, and chronic traumatic encephalopathy. Also, for example, Non-Patent Documents 4 and 5 describe that the structure of α-synuclein amyloid differs between patients with multiple system atrophy, dementia with Lewy bodies, and Parkinson's disease. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Fitzpatrick AWP et al., “Cryo-EM structuresof tau filaments from Alzheimer's disease.”, Nature.2017 Jul 13;547(7662):185-190. [Non-patent document 2] Falcon B, et al. “Structures of filaments from Pick's disease reveal a novel tau protein fold.” Nature. 2018 Sep;561(7721):137-140. [Non-patent document 3] Falcon B, et al., “Novel tau filament fold in chronic traumatic encephalopathyencloses hydrophobic molecules.” Nature. 2019 Apr;568(7752):420-423. [Non-patent document 4] Schweighauser M, et al., “Structures of α-synuclein filaments from multiple system atrophy.” Nature. 2020 Sep;585(7825):464-469. [Non-Patent Document 5] Yang Y, et al., “Structures of α-synuclein filaments from human brains with Lewy pathology.” Nature. 2022 Oct;610(7933):791-795. Summary of the Invention [Problem to be solved by the invention]

[0005] For the reasons described above, the identification of structural polymorphisms of fibrous proteins such as amyloid has attracted attention. Non-Patent Documents 1 to 5 use cryo-electron microscopy to identify structural polymorphisms of amyloid. However, using cryo-electron microscopy to identify structural polymorphisms of amyloid is expensive and requires a long measurement time (approximately 1 to 2 months). Solid-state nuclear magnetic resonance (SMR) can also be used to identify structural polymorphisms of amyloid, but this method is also expensive and requires a long measurement time (approximately several weeks).

[0006] An object of the present invention is to provide a method for easily and quickly identifying structural polymorphisms of fibrous proteins or fibrous peptides. Another object of the present invention is to provide an apparatus and a program that can be used in the method. [Means for solving the problem]

[0007] The present inventors separated the fluorescence decay curve of thioflavin T (hereinafter also referred to as "ThT") bound to amyloid-β fibrils into four exponential components and found that the fluorescence lifetime values ​​of three of these components were derived from ThT bound to different binding sites (in different binding modes) on amyloid-β fibrils. Furthermore, they found that the weighting factors in the exponential components indicate the abundance of different binding sites estimated from the fluorescence lifetime values. Therefore, by measuring the fluorescence lifetime values ​​for each binding site and the weighting factors for each binding site, it is possible to measure slight differences in the structure of fibrous proteins or fibrous peptides as differences in the fluorescence lifetime values ​​and weighting factors.

[0008] The present invention provides a method for identifying structural polymorphisms of a fibrous protein or fibrous peptide, comprising: obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and Thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n (where n is a natural number of 4 or more), The above fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n and identifying a structural polymorphism of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T) among the above.

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[0009] The method of identifying structural polymorphisms of fibrous proteins or fibrous peptides according to the present invention includes the above-described configuration, and therefore can identify structural polymorphisms of fibrous proteins or fibrous peptides simply and quickly.

[0010] In the method for identifying structural polymorphisms of the fibrous protein or fibrous peptide, the exponential fitting comprises convoluting the function G(t) according to the following formula (2) to obtain a function I(t), and comparing the function I(t) with the function F(t) of the fluorescence decay curve to obtain χ in the following formula (3): 2 The method may include searching for a combination of variables that minimizes χ by a nonlinear least squares method. 2 It is desirable that the value be 1.2 or less.

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[0011] In the method for identifying structural polymorphisms of the fibrous protein or fibrous peptide, the identifying step includes: n , and the above weighting factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 associated with the structural polymorphisms of different fibrous proteins or fibrous peptides are n , and weight factors A1 to A n This may be performed by checking against a database in which the structural polymorphisms of fibrous proteins or fibrous peptides are stored.

[0012] In the method for identifying structural polymorphisms of a fibrous protein or fibrous peptide, the exponential fitting may be a four-component exponential fitting.

[0013] In the method for identifying structural polymorphisms of a fibrillar protein or a fibrillar peptide, the protein or peptide forming the fibrillar protein or fibrillar peptide may be an amyloidogenic protein or amyloidogenic peptide.

[0014] In the method for identifying structural polymorphisms of the fibrillar protein or fibrillar peptide, the amyloid-forming protein or peptide may be one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

[0015] The present invention also provides an apparatus for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, comprising: a fluorescence lifetime measurement unit for obtaining a fluorescence decay curve for a sample containing a fibrous protein or a fibrous peptide and thioflavin T; The above fluorescence decay curve is subjected to exponential fitting of four or more components based on G(t) expressed by the following formula (1), and the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n (where n is a natural number equal to or greater than 4), The above fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n and a second calculation unit that identifies structural polymorphisms of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T).

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[0016] In the apparatus for identifying structural polymorphisms of fibrous proteins or fibrous peptides, the exponential fitting comprises convolution integrating the function G(t) according to the following formula (2) to obtain a function I(t), and comparing the function I(t) with the function F(t) of the fluorescence decay curve to obtain χ in the following formula (3): 2 The method may include searching for a combination of variables that minimizes χ by a nonlinear least squares method. 2 It is desirable that the value be 1.2 or less.

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[0017] In the apparatus for identifying structural polymorphisms of fibrous proteins or fibrous peptides, the identification is performed by n , and the above weighting factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 associated with the structural polymorphisms of different fibrous proteins or fibrous peptides are n , and weight factors A1 to A n This may be done by checking against a database in which the following is stored:

[0018] In the apparatus for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, the exponential fitting may be a four-component exponential fitting.

[0019] In the apparatus for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, the protein or peptide forming the fibrous protein or fibrous peptide may be an amyloid-forming protein or amyloid-forming peptide.

[0020] In the device for identifying structural polymorphisms in fibrous proteins or fibrous peptides, the amyloid-forming protein or peptide may be one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

[0021] The present invention also provides a program for identifying structural polymorphisms of fibrous proteins or fibrous peptides, comprising a computer, a fluorescence lifetime measurement unit for obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and thioflavin T, a function F(t) of the fluorescence decay curve, and a function G(t) of four or more components for performing exponential function fitting based on the function G(t) expressed by the following formula (1), to obtain fluorescence lifetime values ​​τ1 to τ2 of each exponential function component. n , and weight factors A1 to A n a first calculation unit that obtains at least one value (where n is a natural number of 4 or more) of each of the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n The present invention also relates to a program for functioning as a second calculation unit that identifies structural polymorphisms of the above-mentioned fibrous protein or fibrous peptide based on the fluorescence lifetime values ​​and weighting factors in the exponential function components derived from the autofluorescence of Thioflavin T (excluding the fluorescence lifetime values ​​and weighting factors in the exponential function components derived from the autofluorescence of Thioflavin T).

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[0022] In the program for identifying structural polymorphisms of the fibrous protein or fibrous peptide, the exponential fitting involves convolution integration of the function G(t) according to the following formula (2) to obtain a function I(t), and comparing the function I(t) with the function F(t) of the fluorescence decay curve to obtain χ in the following formula (3): 2 The method may include searching for a combination of variables that minimizes χ by a nonlinear least squares method. 2 It is desirable that the value be 1.2 or less.

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[0023] In the program for identifying structural polymorphisms of the fibrous protein or fibrous peptide, the identification is performed by n , and the above weighting factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 associated with the structural polymorphisms of different fibrous proteins or fibrous peptides are n , weight factors A1~A n This may be done by checking against a database in which the following is stored:

[0024] In the structural polymorphism identification program for the fibrous protein or fibrous peptide, the exponential fitting may be a four-component exponential fitting.

[0025] In the structural polymorphism identification program for the fibrillar protein or fibrillar peptide, the protein or peptide forming the fibrillar protein or fibrillar peptide may be an amyloid-forming protein or amyloid-forming peptide.

[0026] In the program for identifying structural polymorphisms in fibrous proteins or fibrous peptides, the amyloid-forming protein or peptide may be one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

[0027] The present invention includes, for example, the following inventions. [1] 1. A method for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, comprising: obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and Thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n (where n is a natural number of 4 or more), The above fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n and identifying a structural polymorphism of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T) of the above.

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[10] The apparatus according to any one of [7] to [9], wherein the exponential fitting is a four-component exponential fitting.

[11] The device according to any one of [7] to

[10] , wherein the protein or peptide that forms the fibrous protein or fibrous peptide is an amyloid-forming protein or amyloid-forming peptide.

[12] The device described in

[11] , wherein the amyloid-forming protein or peptide is one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

[13] A structural polymorphism identification program for a fibrous protein or fibrous peptide, Computer, a fluorescence lifetime measurement unit for obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n (where n is a natural number equal to or greater than 4), and The above fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n a second calculation unit that identifies structural polymorphisms of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T) among the above.

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[14] The above exponential fitting is The function G(t) is convoluted according to the following formula (2) to obtain a function I(t); and The above function I(t) is compared with the function F(t) of the fluorescence decay curve, and χ in the following formula (3) is calculated. 2 The program according to

[13] , which includes searching for a combination of variables that minimizes using a nonlinear least squares method.

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[15] The above identification is performed by determining the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 associated with the structural polymorphisms of different fibrous proteins or fibrous peptides are n , weight factors A1~A n The program described in

[13] or

[14] is performed by checking the data against a database stored therein.

[16] The program according to any one of

[13] to

[15] , wherein the exponential fitting is four-component exponential fitting.

[17] The program according to any one of

[13] to

[16] , wherein the protein or peptide that forms the fibrous protein or fibrous peptide is an amyloid-forming protein or amyloid-forming peptide.

[18] The program according to

[17] , wherein the amyloid-forming protein or peptide is one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof. [Effects of the Invention]

[0028] According to the present invention, a method for easily and quickly identifying structural polymorphisms of a fibrous protein or a fibrous peptide can be provided. The present invention also provides an apparatus and a program that can be used in the method. [Brief explanation of the drawings]

[0029] [Figure 1] FIG. 1 is a schematic diagram showing the hardware configuration of a structural polymorphism identification device D for a fibrous protein or fibrous peptide. [Figure 2] FIG. 1 is a schematic diagram showing the functional configuration of a structural polymorphism discrimination device D for a fibrous protein or fibrous peptide. [Figure 3] Fluorescence decay curves for a sample containing amyloid β fibrils and ThT. [Figure 4] Graph (A) shows the fluorescence intensity of each exponential function component obtained when amyloid beta fibrils were analyzed using a measurement sample prepared at an amyloid beta concentration of 2 μM, plotted against the molar concentration ratio of amyloid beta fibrils to ThT ([ThT] / [amyloid beta]). Graph (B) shows the fluorescence intensity of each exponential function component obtained when amyloid beta fibrils were analyzed using a measurement sample prepared at an amyloid beta concentration of 0.8 μM, plotted against the molar concentration ratio of amyloid beta fibrils to ThT ([ThT] / [amyloid beta]). [Figure 5]Graph (A) shows the results of comparing the fluorescence lifetime values ​​in the exponential function component C3 between amyloid β(1-40) fibrils and amyloid β(1-42) fibrils, and Graph (B) shows the results of comparing the fluorescence lifetime values ​​in the exponential function component C4 between amyloid β(1-40) fibrils and amyloid β(1-42) fibrils. [Figure 6] Graph (A) shows the results of comparing the fluorescence lifetime values ​​in the exponential function component C3 between amyloid β(1-42) fibrils formed under different conditions, and graph (B) shows the results of comparing the fluorescence lifetime values ​​in the exponential function component C4 between amyloid β(1-42) fibrils formed under different conditions. DETAILED DESCRIPTION OF THE INVENTION

[0030] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following describes in detail the embodiments of the present invention, but the present invention is not limited to the following embodiments.

[0031] Method for distinguishing structural polymorphisms of fibrous proteins or fibrous peptides The method for identifying structural polymorphisms of a fibrous protein or a fibrous peptide according to the present embodiment (hereinafter also referred to as the "identification method according to the present embodiment") includes the steps of: obtaining a fluorescence decay curve for a sample containing a fibrous protein or a fibrous peptide and ThT (measurement step); fitting a function F(t) of the fluorescence decay curve with an exponential function of four or more components based on a function G(t) expressed by the following formula (1); and obtaining fluorescence lifetime values ​​τ1 to τ2 of each exponential function component. n , and weight factors A1 to A n (where n is a natural number of 4 or more) (fitting step), n , and the above weighting factors A1 to A n The method includes a step of identifying the structural polymorphism of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of ThT) (identification step).

[0032]

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[0033] As used herein, "structural polymorphism of a fibrous protein or fibrous peptide" means that a fibrous protein or fibrous peptide formed from a single type of peptide or protein (a peptide or protein having the same amino acid sequence) has different structures.

[0034] (Measurement process) The measurement step is a step of obtaining a fluorescence decay curve for a sample containing a fibrous protein or a fibrous peptide and ThT. In the measurement step, the sample containing the fibrous protein or the fibrous peptide and ThT is irradiated with excitation light that excites ThT, and the fluorescence decay of the sample containing the fibrous protein or the fibrous peptide and ThT is measured.

[0035] The sample containing a fibrous protein or fibrous peptide and ThT is not particularly limited as long as it contains these, and can be prepared by mixing a sample containing a fibrous protein or fibrous peptide with ThT. The sample containing a fibrous protein or fibrous peptide may or may not be a biological sample. Examples of biological samples containing a fibrous protein or fibrous peptide include brain slices, cerebrospinal fluid, blood, and mucosa. The sample containing a fibrous protein or fibrous peptide may be a solution obtained by suspending or dissolving these samples in an appropriate medium (e.g., water, buffer solution, culture medium, etc.), or may be a solution obtained by filtering insoluble matter after suspension or dissolution. The sample containing a fibrous protein or fibrous peptide and ThT may be in the form of a liquid or solid, but is preferably a liquid.

[0036] As used herein, a "fibrous protein or fibrous peptide" refers to a protein or peptide assembly formed by the assembly of protein or peptide molecules into a fibrous shape. The fibrous protein or fibrous peptide may be, for example, amyloid. "Amyloid" refers to a special insoluble aggregate (fibrous protein or fibrous peptide) of a protein or peptide characterized by a β-sheet structure. The protein or peptide forming the fibrous protein or fibrous peptide is not particularly limited, and may be, for example, an amyloid-forming protein or amyloid-forming peptide. The amyloid-forming protein or amyloid-forming peptide is preferably one or more selected from the group consisting of amyloid β, α-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, β2-microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof, and more preferably one or more selected from the group consisting of amyloid β, α-synuclein, tau, and partial peptides thereof. The protein or peptide forming the fibrous protein or fibrous peptide may be, for example, collagen, keratin, elastin, fibroin, actin, myosin, fibronectin, laminin, and the like.

[0037] Thioflavin T (ThT) is a known compound represented by the following formula, also known as 4-(3,6-dimethyl-1,3-benzothiazol-3-ium-2-yl)-N,N-dimethylaniline chloride. ThT is a fluorescent dye widely used for staining amyloid (ThT staining).

[0038] [ka]

[0039] In the identification method according to this embodiment, for example, commercially available ThT reagents can be used without any particular restrictions.

[0040] The concentration of the fibrous protein or fibrous peptide in a sample containing a fibrous protein or fibrous peptide and ThT may be, for example, 0.2 μM or more, 0.5 μM or more, 0.8 μM or more, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, or 5 μM or more, or may be 20 μM or less, 15 μM or less, or 10 μM or less, when the sample is liquid. The concentration of ThT in a sample containing a fibrous protein or fibrous peptide and ThT may be, for example, 0.2 μg / g or more, 0.4 μg / g or more, 0.6 μg / g or more, 0.8 μg / g or more, 1 μg / g or more, or 1.5 μg / g or more when the sample is solid, or may be 10 μg / g or less, 9 μg / g or less, 8 μg / g or less, 7 μg / g or less, 6 μg / g or less, 5 μg / g or less, 4 μg / g or less, or 3 μg / g or less.

[0041] The concentration of ThT in a sample containing a fibrous protein or fibrous peptide and ThT may be, for example, 1 μM or more, 2 μM or more, 3 μM or more, 4 μM or more, or 5 μM or more when the sample is liquid, or may be 20 μM or less, 15 μM or less, or 10 μM or less. For example, the concentration of ThT in a sample containing a fibrous protein or fibrous peptide and ThT may be, for example, 0.2 μg / g or more, 0.4 μg / g or more, 0.6 μg / g or more, 0.8 μg / g or more, 1 μg / g or more, or 1.5 μg / g or more when the sample is solid, or may be 10 μg / g or less, 9 μg / g or less, 8 μg / g or less, 7 μg / g or less, 6 μg / g or less, 5 μg / g or less, 4 μg / g or less, or 3 μg / g or less.

[0042] In a sample containing a fibrous protein or fibrous peptide and ThT, the ratio of the molar concentration of ThT to the molar concentration of the protein or peptide that forms the fibrous protein or fibrous peptide ([ThT] / [protein or peptide that forms the fibrous protein or fibrous peptide]) may be, for example, greater than 0, 0.01 or more, 0.05 or more, 0.1 or more, 0.5 or more, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more. In a sample containing a fibrous protein or fibrous peptide and ThT, the ratio of the molar concentration of ThT to the molar concentration of the protein or peptide that forms the fibrous protein or fibrous peptide ([ThT] / [protein or peptide that forms the fibrous protein or fibrous peptide]) may be, for example, 100 or less, 95 or less, 90 or less, 85 or less, 80 or less, 75 or less, 70 or less, 65 or less, 60 or less, 55 or less, 50 or less, 40 or less, or 30 or less. The molar concentration of the protein or peptide that forms the fibrous protein or fibrous peptide is the molar concentration converted into a protein or peptide (e.g., amyloid β) that can form the fibrous protein or fibrous peptide.

[0043] The fluorescence decay of a sample containing a fibrous protein or fibrous peptide and ThT can be measured by a known method. Specifically, for example, a fluorescence decay curve can be obtained by irradiating a sample containing a fibrous protein or fibrous peptide and ThT with light having a wavelength of 280 to 800 nm, 300 to 600 nm, 350 to 500 nm, or 400 to 420 nm (preferably 412 nm) using a fluorescence lifetime measurement device, and detecting the fluorescence emitted from the sample over time with a wavelength of 400 to 600 nm, 420 to 580 nm, or 450 to 550 nm.

[0044] (Fitting process) In the fitting step, the function F(t) of the fluorescence decay curve obtained in the measurement step is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime values ​​τ1 to τ2 of each of the above exponential function components are calculated. n , and the above weighting factors A1 to A n (where n is a natural number of 4 or more).

[0045]

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[0046] The fluorescence expression of ThT bound to a fibrous protein or peptide is thought to be affected by the restriction of the free rotation of the benzothiazole ring relative to the aminobenzene ring of ThT upon binding to the fibrous protein or peptide (Vitali I. et al., J. Phys. Chem. B 2008, 112, 49, 15893-15902). The strength of the bond between ThT and the fibrous protein or peptide is thought to affect the restriction of free rotation. Therefore, the fluorescence lifetime values ​​of ThT for each of the above exponential components are thought to reflect the strength of the bond between ThT and the fibrous protein or peptide (i.e., differences in the binding site (binding mode) to the fibrous protein or peptide).

[0047] For this reason, the fluorescence lifetime values ​​τ1 to τ2 of the above exponential function components obtained by the fitting process are nis considered to reflect the difference in the binding site of ThT on the fibrous protein or fibrous peptide, excluding the fluorescence lifetime value in the exponential function component derived from the autofluorescence of ThT. Furthermore, the weighting factor of each of the above exponential function components is considered to reflect the abundance of different binding sites corresponding to the above fluorescence lifetime value, excluding the weighting factor in the exponential function component derived from the autofluorescence of ThT. Therefore, based on these values, structural polymorphisms of fibrous proteins or fibrous peptides can be identified in the identification step described below. Note that the fluorescence lifetime value in the exponential function component derived from the autofluorescence of ThT is the fluorescence lifetime value derived from ThT not bound to the fibrous protein or fibrous peptide, and the obtained fluorescence lifetime values ​​τ1 to τ n The weighting factor for the exponential component resulting from the autofluorescence of ThT is combined with the fluorescence lifetime value for the exponential component resulting from the autofluorescence of ThT.

[0048] The fitting step may involve, for example, convoluting the function G(t) according to the following formula (2) to obtain a function I(t), and comparing the function I(t) with the function F(t) of the fluorescence decay curve to obtain χ in the following formula (3): 2 The variables that minimize n , A1~A n ) by a nonlinear least squares method. n , A1~A n The best combination of

[0049] The results of this analysis are n are the lifetime values ​​of each fluorescent component, and A1 to A n indicates the weight factor of each fluorescent component, i.e., the amount of the fluorescent component.

[0050]

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[0051]

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[0052] Here, the value of n is the number of exponential functions required to analyze the fluorescence decay curve, and each component originates from a different physical mechanism and / or fluorescence component. The value of n can be any value, but χ 2 The number of components is preferably set to a value sufficient to obtain good fitting, with a target value of ≦1.2 as a guideline, and more preferably set to 4. In this embodiment, these components originate from fluorescent components derived from the complex of a fibrous protein or fibrous peptide with ThT, fluorescent components due to the autofluorescence of ThT, apparent fluorescent components derived from the characteristics of the instrument used to measure the fluorescence lifetime, fluorescent components derived from impurities contained in the sample, etc.

[0053] If there is a noise component, the noise parameter (τ n+1 ~τ m , A n+1 ~A m The fitting step may be performed by calculating G(t) expressed by the following formula (4), which includes G(t) expressed by the formula (1) above.

[0054]

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[0055] (Identification process) The identification process uses the fluorescence lifetime values ​​τ1 to τ2 obtained in the fitting process. n , and weight factors A1 to A n and (b) identifying structural polymorphisms of fibrous proteins or fibrous peptides based on at least one of the above (excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of ThT).

[0056] Identifying structural polymorphisms of a fibrous protein or fibrous peptide may be, for example, estimating structural polymorphisms of a fibrous protein or fibrous peptide of interest, or determining whether structural polymorphisms of two fibrous proteins or fibrous peptides of interest are identical, etc.

[0057] In the identification step, when a structural polymorphism of a fibrous protein or fibrous peptide of interest is estimated, for example, the identification step may be carried out by using the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 associated with the structural polymorphisms of different fibrous proteins or fibrous peptides are n , and weight factors A1 to A n The fluorescence lifetime values ​​τ1 to τ2 obtained in the fitting step may be compared with a database in which the values ​​are stored. n , and weight factors A1 to A n and the fluorescence lifetime values ​​τ1 to τ stored in the database. n , and weight factors A1 to A n In comparison with the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n If there is no significant difference in at least one of the fluorescence lifetime values ​​τ1 to τ2 stored in the database, the two structural polymorphisms may be determined to be identical. n , and weight factors A1 to A nIt can be determined that the structural polymorphism is the same as the corresponding structural polymorphism. For example, if both τ1 and A1 obtained in the fitting step are not significantly different from the comparison target, it can be determined that the structural polymorphisms are the same. Note that the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n and the fluorescence lifetime values ​​τ1 to τ stored in the database. n , and weight factors A1 to A n In comparison with the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n For example, even if τ1 obtained in the fitting step is not significantly different from the comparison target, if A1 obtained in the fitting step is significantly different from the comparison target, it may be determined that the two structural polymorphs are different.

[0058] The database can be created, for example, as follows: First, structural polymorphisms of various fibrous proteins or fibrous peptides are analyzed in advance using a cryo-electron microscope or the like to obtain structural polymorphism data for each fibrous protein or fibrous peptide. Then, the identification method according to this embodiment is performed on fibrous proteins or fibrous peptides having the same structural polymorphism as the structural polymorphism of each fibrous protein or fibrous peptide to obtain fluorescence lifetime values ​​τ1 to τ2. n , weight factors A1~A n Next, the structural polymorphism data of the fibrous protein or fibrous peptide and the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n and are associated.

[0059] In addition, in the identification step, when determining whether the structural polymorphisms of two fibrous proteins or fibrous peptides of interest are identical, the fluorescence lifetime values ​​τ1 to τ n , and weight factors A1 to A n This can be done by comparing

[0060] In the identification process, the fluorescence lifetime values ​​τ1 to τ2 obtained in the fitting process are n , and weight factors A1 to A n It is sufficient to use at least one of the above fluorescence lifetime values ​​τ1 to τ2 (excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of ThT). n , and the above weighting factors A1 to A n may be based on at least two or more, three or more, four or more, or all of the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n It may be based on 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% (all) of each of the above.

[0061] [Device for identifying structural polymorphisms of fibrous proteins or fibrous peptides] The apparatus for identifying structural polymorphisms in a fibrous protein or fibrous peptide according to this embodiment (hereinafter also referred to as the "apparatus according to this embodiment") includes a fluorescence lifetime measurement unit that obtains a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and thioflavin T, and performs exponential function fitting of four or more components based on a function G(t) expressed by the following formula (1) to the function F(t) of the fluorescence decay curve, thereby obtaining fluorescence lifetime values ​​τ1 to τ2 of each exponential function component. n , and weight factors A1 to A n (where n is a natural number of 4 or more), and a first calculation unit that obtains the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n and a second calculation unit that identifies the structural polymorphism of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of ThT).

[0062]

number

[0063] FIG. 1 is a schematic diagram showing the hardware configuration of a structural polymorphism identification device D for a fibrous protein or fibrous peptide according to one embodiment, and FIG. 2 is a schematic diagram showing the functional configuration of a structural polymorphism identification device D for a fibrous protein or fibrous peptide according to one embodiment.

[0064] 1 , the apparatus D for identifying structural polymorphisms in a fibrous protein or fibrous peptide is physically configured as a conventional computer including a CPU D11, main storage devices such as a ROM D12 and RAM D13, input devices D14 such as a keyboard and a mouse, output devices D15 such as a display, a communication module D16 such as a network card for transmitting and receiving data to and from other devices such as an imaging device C, and an auxiliary storage device D17 such as a hard disk. Each function of the apparatus D for identifying structural polymorphisms in a fibrous protein or fibrous peptide, which will be described later, is realized by loading predetermined computer software onto hardware such as the CPU D11, ROM D12, and RAM D13, thereby operating the input device D14, output device D15, and communication module D16 under the control of the CPU D11, and reading and writing data from and to the main storage devices D12 and D13 and the auxiliary storage device D17.

[0065] As shown in Figure 2, the structural polymorphism identification device D for a fibrous protein or fibrous peptide of one embodiment has, as functional components, a fluorescence lifetime measurement unit D1, a first calculation unit D2, a second calculation unit D3, and a display unit D4.

[0066] The fluorescence lifetime measurement unit D1 acquires fluorescence decay curve data obtained by a fluorescence lifetime measurement device (not shown). The first calculation unit D2 performs exponential function fitting of four or more components based on the function G(t) expressed by the above formula (1) to the fluorescence decay curve from the acquired fluorescence decay curve data, and calculates the fluorescence lifetime values ​​τ1 to τ2 of each of the above exponential function components. n , and weight factors A1 to A n(where n is a natural number equal to or greater than 4). The second calculation unit D3 calculates the fluorescence lifetime values ​​τ1 to τ n , and the above weighting factors A1 to A n The structural polymorphism of the fibrous protein or fibrous peptide is identified based on the fluorescence lifetime values ​​and weighting factors in the exponential function components derived from the autofluorescence of ThT (excluding the fluorescence lifetime values ​​and weighting factors in the exponential function components derived from the autofluorescence of ThT). Display unit D4 displays the identification results. The fibrous protein or fibrous peptide is as described above, and the fluorescence lifetime measurement unit can apply the aspects described in the measurement step, the first calculation unit can apply the aspects described in the fitting step, and the second calculation unit can apply the aspects described in the identification step.

[0067] [Program for identifying structural polymorphisms of fibrous proteins or fibrous peptides] The program for identifying structural polymorphisms in a fibrous protein or fibrous peptide causes a computer to function as the above-described fluorescence lifetime measurement unit D1, first calculation unit D2, second calculation unit D3, and display unit D4. By loading the program for identifying structural polymorphisms in a fibrous protein or fibrous peptide into a computer, the computer operates as the device D for identifying structural polymorphisms in a fibrous protein or fibrous peptide. The program for identifying structural polymorphisms in a fibrous protein or fibrous peptide is provided, for example, by being recorded on a computer-readable recording medium. The recording medium may be a non-transitory recording medium. Examples of the recording medium include recording media such as flexible disks, CDs, and DVDs, recording media such as ROMs, and semiconductor memories. The fibrous protein or fibrous peptide is as described above, and the fluorescence lifetime measurement unit can be configured as described in the measurement step, the first calculation unit can be configured as described in the fitting step, and the second calculation unit can be configured as described in the identification step. [Example]

[0068] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0069] [Test Example 1: Fluorescence lifetime analysis of thioflavin T in amyloid β fibrils] (1. Preparation of measurement sample) Amyloid β protein (trade name: Amyloid β Protein Human 1-42, manufactured by Peptide Institute) was dissolved in dimethyl sulfoxide to a concentration of 5 mM (initial dissolution), and then further diluted with 10 mM hydrochloric acid to a concentration of 100 μM. The resulting amyloid β protein preparation was statically cultured at 37°C using a shaking incubator (manufactured by AS ONE Corporation) to prepare a sample containing amyloid β fibrils (hereinafter also referred to as the "amyloid β fibril sample"). Next, 10 μL of the amyloid β fibril sample was mixed with 990 μL of a 5 μM ThT solution (solvent: 50 mmol / L glycine-sodium hydroxide solution, pH 9.0) to prepare a measurement sample.

[0070] (2. Acquisition of Fluorescence Decay Curve) The measurement samples obtained in (1. Preparation of Measurement Samples) were each dispensed into quartz cells. Then, a fluorescence decay curve was obtained using a fluorescence lifetime measurement device (manufactured by Hamamatsu Photonics, trade name Quantaurus-Tau, light source: pulsed laser diode PLP-10 (manufactured by Hamamatsu Photonics)) with an excitation wavelength of 405 nm, an observation wavelength of 500 nm, and a time range of 20 ns (time resolution 0.020 ns). The results are shown in Figure 3.

[0071] (3. Four-component exponential fitting of fluorescence decay curves) For the fluorescence decay curve F(t) obtained in (2. Obtaining the Fluorescence Decay Curve), multi-component exponential fitting was performed using the fluorescence lifetime value τ and the weighting factor A as variables, according to the following procedure, in accordance with the non-patent literature (DVO'Cornor, "Nano-Picosecond Fluorescence Measurement and Analysis Method - Time-Correlated Single Photon Counting Method", Academic Press, Chapter 2, pp. 33-51, Chapter 6, pp. 153-206).

[0072] First, the function G(t) expressed by Equation (1) was convoluted according to Equation (2) to obtain the function I(t). Next, the above I(t) was compared with the measured fluorescence decay curve F(t), and the χ in Equation (3) was adjusted so that the two functions best matched. 2 The combination of variables that minimizes the value of 1.2 or less was searched for using the nonlinear least squares method.

[0073]

number

[0074]

number

[0075]

number

[0076] The above multi-component exponential fitting revealed that the fluorescence decay curve obtained in (2. Obtaining the Fluorescence Decay Curve) above required four exponential components C1 to C4, and the exponential components τ1 to τ4 and A1 to A4 of C1 to C4 were obtained. τ1 to τ4 of C1 to C4 are shown in Table 1.

[0077] [Table 1]

[0078] τ1 has the shortest fluorescence lifetime and is attributed to ThT not bound to amyloid β fibrils. τ2 to τ4 are attributed to ThT bound to amyloid β fibrils. To examine τ2 to τ4 in more detail, we analyzed the changes in the fluorescence intensity behavior of the exponential components of C2 to C4 with increasing ThT concentration.

[0079] The fluorescence intensity of each exponential function component was calculated using the following formula (5) when the amyloid-β concentration was 2 μM and the ThT concentration relative to the amyloid-β concentration ([ThT] / [Amyloid-β]) was 0.15, 0.29, 0.59, 1.2, or 2.3. The fluorescence intensity of each exponential function component was then plotted against [ThT] / [Amyloid-β]. A graph showing the change in fluorescence intensity with ThT concentration was also obtained under conditions with higher ThT concentrations, as described above. The fluorescence intensity of each exponential function component was also calculated using the following formula (5) when the amyloid-β concentration was 0.8 μM and the ThT concentration relative to the amyloid-β concentration ([ThT] / [Amyloid-β]) was 0.91, 1.8, 3.7, 7.3, or 14.6. The fluorescence intensity of each exponential function component was then plotted against [ThT] / [Amyloid-β]. The results are shown in Figures 4(A) and 4(B).

[0080]

number

[0081] As shown in Figure 4(A) and (B), the behavior of the C2 to C4 exponential components varied depending on the ThT concentration. Furthermore, the fluorescence intensity of C4, determined by τ4 with the highest fluorescence lifetime, first saturated and then decreased with increasing ThT concentration.

[0082] Regarding amyloid-β fibrils and ThT, some reports suggest the existence of multiple ThT binding sites on amyloid-β fibrils (Chun Wu et al., Biophys J. 2011 Mar 2; 100(5): 1316-1324., Andrew Lockhart et al., J Biol Chem. 2005 Mar 4;280(9):7677-84). Based on these reports and the above results, τ2–τ4 are considered to be the fluorescence lifetimes of ThT bound to different binding sites on amyloid-β fibrils.

[0083] Therefore, by measuring the fluorescence lifetime of each ThT bound to a fibrous protein or fibrous peptide such as amyloid beta fibrils for each binding site, slight differences in the structure of the fibrous protein or fibrous peptide can be measured as differences in each fluorescence lifetime, and structural polymorphisms of the fibrous protein or fibrous peptide can be identified.

[0084] [Test Example 2: Structural discrimination between amyloid β(1-40) fibrils and amyloid β(1-42) fibrils] (1. Preparation of measurement sample) Amyloid β protein (trade names: Amyloid β Protein Human 1-40 and Amyloid β Protein Human 1-42, manufactured by Peptide Institute) was dissolved in 10 mM sodium hydroxide to a concentration of 1 mg / mL and further diluted 6-fold with 50 mM phosphate buffer, pH 7.4. The resulting amyloid β protein preparation was incubated at 37°C with shaking (agitation: 800 rpm) in a shaking incubator (manufactured by AS ONE Corporation) to prepare a sample containing amyloid β fibrils. Subsequently, the amyloid β fibril sample (final concentration 2 μM), a ThT aqueous solution (final concentration 4 μM), and 50 mM phosphate buffer, pH 7.4 were mixed to prepare a measurement sample.

[0085] (2. Acquisition of Fluorescence Decay Curve) In the same manner as in Test Example 1, the fluorescence decay curve was measured.

[0086] (3. Four-component exponential fitting of fluorescence decay curves) Four-component exponential fitting was performed on each of the obtained fluorescence decay curves F(t) in the same manner as in Test Example 1. τ1 to τ4 and A1 to A4 were obtained by performing the above fitting. τ1 to τ4 and A1 to A4 are shown in Table 2. Furthermore, the results of comparing the fluorescence lifetime values ​​of C3 (τ3) and C4 (τ4) in amyloid β(1-40) fibrils and amyloid β(1-42) fibrils are shown in Figures 5(A) and 5(B).

[0087] [Table 2]

[0088] As shown in Table 2 and Figures 5(A)-(B), there were significant differences in τ3 and τ4 between amyloid β(1-40) fibrils and amyloid β(1-42) fibrils. The amino acid sequences of amyloid β(1-40) and amyloid β(1-42) fibrils are different, and the structures of amyloid β fibrils are also different. By measuring multiple fluorescence lifetimes of each amyloid β fibril, we were able to determine that the structures were different.

[0089] [Test Example 3: Identification of structural polymorphisms of amyloid β fibrils] It is known that amyloid β(1-42) forms amyloid fibrils with different structural polymorphisms when the culturing conditions are changed. In Test Example 3, amyloid β fibrils with different structures were formed to investigate whether amyloid proteins with the same amino acid sequence could be distinguished.

[0090] (1. Preparation of measurement sample) Amyloid β (1-42) fibril samples were prepared in the same manner as in Test Example 1, except that the conditions were changed as shown in Table 3 below. Subsequently, measurement samples (Samples 1 to 3) were prepared using each of the prepared amyloid β fibril samples in the same manner as in Test Example 1, except that the compositions were changed as shown in Table 3 below.

[0091] [Table 3]

[0092] (2. Acquisition of Fluorescence Decay Curve) In the same manner as in Test Example 1, the fluorescence decay curve was measured.

[0093] (3. Four-component exponential fitting of fluorescence decay curves) Four-component exponential fitting was performed on each of the obtained fluorescence decay curves F(t) in the same manner as in Test Example 1. By performing the above fitting, τ1 to τ4 and A1 to A4 in Equation (1) were obtained. The results are shown in Table 4. Furthermore, the results of comparing the fluorescence lifetime value of C3 (τ3) or the fluorescence lifetime value of C4 (τ4) for each sample are shown in Figures 6(A) and 6(B). Furthermore, the ratio (%) of A2 to the total value of A1 to A4 for each sample is shown in Table 5.

[0094] [Table 4]

[0095] [Table 5]

[0096] As shown in Table 4 and Figures 6(A)-(B), there was a significant difference between the τ3 and τ4 values ​​obtained from each sample. Furthermore, as shown in Table 4, there was no significant difference between the τ2 values ​​obtained from each sample. This suggests that similar binding sites exist in each sample. On the other hand, as shown in Table 5, A2 relative to the total values ​​of A1-A4 in each sample differed significantly between samples. This suggests that the difference in weighting factors indicates that the abundance ratio of ThT binding sites, as estimated from the τ2 values, differs between structural polymorphs. These results demonstrate that structural polymorphs of amyloid beta fibrils with the same amino acid sequence can be distinguished by measuring multiple fluorescence lifetime values ​​and weighting factors.

[0097] From the results of Test Examples 1 to 3, it is believed that by measuring the fluorescence lifetime value for each binding site and measuring the weighting factor for each abundance of that binding site, slight differences in the structure of fibrous proteins or fibrous peptides can be measured as differences in each fluorescence lifetime value and each weighting factor. [Explanation of symbols]

[0098] D... structural polymorphism identification device for fibrous protein or fibrous peptide, D1... fluorescence lifetime measurement unit, D2... first calculation unit, D3... second calculation unit, D4... display unit, D11... CPU, D12... ROM, D13... RAM, D14... input device, D15... output device, D16... communication module, D17... auxiliary storage device.

Claims

1. 1. A method for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, comprising: obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and Thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime value τ of each exponential function component is calculated. １ ~τ ｎ , and weight factor A １ ~A ｎ (where n is a natural number of 4 or more), The fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ and identifying a structural polymorphism of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T) of the above. [Equation 1] [In formula (1), n ​​represents a natural number of 4 or more, and A ｉ denotes the weight factor of each exponential function component, t denotes a variable representing time, and τ ｉ indicates the fluorescence lifetime value of each exponential function component.]

2. The exponential fitting The function G(t) is subjected to convolution integration according to the following formula (2) to obtain a function I(t); and The function I(t) is compared with the function F(t) of the fluorescence decay curve, and χ in the following formula (3) is obtained. ２ 2. The method of claim 1, further comprising searching for a combination of variables that minimizes .times. ... [Equation 2] [In formula (2), E(t) represents the instrument response function of the fluorescence lifetime measurement device, C represents the background, and t and t′ represent variables representing time.] [Equation 3] [In formula (3), t ｊ denotes a variable representing time, and p １ indicates the start time of the analysis, and p ２ indicates the end time of the analysis.]

3. The identifying step is performed by determining the fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ , and the fluorescence lifetime values ​​τ associated with each of the different amyloid structural polymorphs. １ ~τ ｎ , and weight factor A １ ~A ｎ The method of claim 1 , wherein the method is performed by checking against a database in which the following is stored:

4. The method of claim 1 , wherein the exponential fit is a four-component exponential fit.

5. The method according to any one of claims 1 to 4, wherein the protein or peptide that forms the fibrillar protein or fibrillar peptide is an amyloidogenic protein or amyloidogenic peptide.

6. 6. The method of claim 5, wherein the amyloidogenic protein or amyloidogenic peptide is one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

7. An apparatus for identifying structural polymorphisms of a fibrous protein or a fibrous peptide, a fluorescence lifetime measurement unit for obtaining a fluorescence decay curve for a sample containing a fibrous protein or a fibrous peptide and thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime value τ of each exponential function component is calculated. １ ~τ ｎ , and weight factor A １ ~A ｎ (where n is a natural number equal to or greater than 4), The fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ and a second calculation unit that identifies structural polymorphisms of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T). [Equation 4] [In formula (1), n ​​represents a natural number of 4 or more, and A ｉ denotes the weight factor of each exponential function component, t denotes a variable representing time, and τ ｉ indicates the fluorescence lifetime value of each exponential function component.]

8. The exponential fitting The function G(t) is subjected to convolution integration according to the following formula (2) to obtain a function I(t); and The function I(t) is compared with the function F(t) of the fluorescence decay curve, and χ in the following formula (3) is obtained. ２ The apparatus according to claim 7, further comprising searching for a combination of variables that minimizes the value of .times. ... [Equation 5] [In formula (2), E(t) represents the instrument response function of the fluorescence lifetime measurement device, C represents the background, and t and t′ represent variables representing time.] [Equation 6] [In formula (3), t ｊ denotes a variable representing time, and p １ indicates the start time of the analysis, and p ２ indicates the end time of the analysis.]

9. The identifying step determines the fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ , and the fluorescence lifetime values ​​τ associated with each of the different amyloid structural polymorphs. １ ~τ ｎ , and weight factor A １ ~A ｎ The apparatus of claim 7, wherein the above is performed in accordance with a database in which the above is stored.

10. The apparatus of claim 7 , wherein the exponential fit is a four-component exponential fit.

11. The device according to any one of claims 7 to 10, wherein the protein or peptide that forms the fibrillar protein or fibrillar peptide is an amyloid-forming protein or amyloid-forming peptide.

12. The device of claim 11, wherein the amyloid-forming protein or peptide is one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

13. A structural polymorphism identification program for a fibrous protein or fibrous peptide, Computer, a fluorescence lifetime measurement unit for obtaining a fluorescence decay curve for a sample containing a fibrous protein or fibrous peptide and thioflavin T; The function F(t) of the fluorescence decay curve is subjected to exponential fitting of four or more components based on the function G(t) expressed by the following formula (1), and the fluorescence lifetime value τ of each exponential function component is calculated. １ ~τ ｎ , and weight factor A １ ~A ｎ (where n is a natural number equal to or greater than 4), and The fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ a second calculation unit that identifies structural polymorphisms of the fibrous protein or fibrous peptide based on at least one of the values ​​(excluding the fluorescence lifetime value and weighting factor in the exponential function component derived from the autofluorescence of Thioflavin T) among the above. [Equation 7] [In formula (1), n ​​represents a natural number of 4 or more, and A ｉ denotes the weight factor of each exponential function component, t denotes a variable representing time, and τ ｉ indicates the fluorescence lifetime value of each exponential function component.]

14. The exponential fitting The function G(t) is subjected to convolution integration according to the following formula (2) to obtain a function I(t); and The function I(t) is compared with the function F(t) of the fluorescence decay curve, and χ in the following formula (3) is obtained. ２ 14. The program according to claim 13, further comprising searching for a combination of variables that minimizes the above by a nonlinear least squares method. [Equation 8] [In formula (2), E(t) represents the instrument response function of the fluorescence lifetime measurement device, C represents the background, and t and t′ represent variables representing time.] [Equation 9] [In formula (3), t ｊ denotes a variable representing time, and p １ indicates the start time of fluorescence decay, and p ２ indicates the end time of the analysis.]

15. The identifying step determines the fluorescence lifetime value τ １ ~τ ｎ , and the weighting factor A １ ~A ｎ , and the fluorescence lifetime values ​​τ associated with each of the different amyloid structural polymorphs. １ ~τ ｎ , and weight factor A １ ~A ｎ The program according to claim 13, wherein the program is executed by checking against a database in which the above-mentioned items are stored.

16. The program of claim 13 , wherein the exponential fitting is a four-component exponential fitting.

17. The program according to any one of claims 13 to 16, wherein the protein or peptide that forms the fibrillar protein or fibrillar peptide is an amyloid-forming protein or amyloid-forming peptide.

18. The program according to claim 17, wherein the amyloid-forming protein or peptide is one or more selected from the group consisting of amyloid beta, alpha-synuclein, transactivation response DNA-binding protein-43, superoxide dismutase 1, prion protein, beta-2 microglobulin, immunoglobulin light chain protein, transthyretin, tau, and partial peptides thereof.

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