JP7499503B2 - Method, device and program for evaluating protein structural changes - Google Patents

Description

The present invention relates to a method for evaluating structural changes in proteins, and belongs to the field of industrial use of proteins.

Circular dichroism and fluorescence spectra are known to sensitively reflect changes in the higher-order structure of proteins, and these measurements play an important role as methods for evaluating structural changes in proteins in the industrial application of proteins, including biopharmaceuticals.

Protein structures are broadly divided into primary, secondary, tertiary, and quaternary structures.
Primary structure refers to the amino acid sequence of a protein.
Secondary structure refers to the partial three-dimensional structure (alpha helix and beta sheet) of the primary protein structure.
Tertiary structure refers to the three-dimensional conformation that a protein molecule takes when the protein secondary structures are spatially organized.
Quaternary structure refers to the state in which multiple polypeptide chains are assembled to form a complex protein.
In the following, existing methods for evaluating changes in the higher-order structure of proteins using CD spectra and fluorescence spectra (References 1 to 3) will be described.

<Circular dichroism measurement>
Reference 1 is an example of measuring the CD spectrum of poly-L-glutamic acid. It is known that poly-L-glutamic acid undergoes an "α-helix-random coil transition" in its secondary structure as the pH changes in an aqueous solution. Looking at Figure 3 (CD of polypeptide) in Reference 1, the CD spectra of poly-L-glutamic acid at pH 4.3 and pH 11 are displayed in the wavelength range of 400 to 200 nm.

According to the CD spectrum in Reference 1, at pH 4.3, a negative Cotton effect appears around 225 nm, and the magnitude of this peak is used as an indicator of the α-helix content. Also, a strong positive Cotton effect appears in the short wavelength region below 200 nm. On the other hand, at pH 11, poly-L-glutamic acid exists in a random coil state, so the above Cotton effect changes significantly.

In this way, it has been explained that changes in the secondary structure of a protein can be estimated based on changes in the CD intensity in a specific wavelength range in the CD spectrum.

<Fluorescence measurement>
Fluorescence measurement can be used as a method for evaluating changes in the tertiary structure of proteins. For example, aromatic amino acids (tryptophan, tyrosine, and phenylalanine) present in protein molecules are often used as fluorescent probes.

Reference 2 introduces a method for evaluating the denaturation process of protein three-dimensional structure when physical parameters such as pH and pressure are changed, based on such amino acid-derived fluorescence spectra. Figure 1 (left) in Reference 2 shows the fluorescence spectrum measured by changing the pH condition of egg white allergen protein from "4 → 10.4 → 12" in that order. In this figure, the peak wavelength of the fluorescence spectrum shifts from 304 nm (fluorescence derived from tyrosine) to 340 nm (fluorescence derived from tyrosinate). From this, it is assessed that H+ dissociates from the side chain OH group of tyrosine as the pH changes to the basic side, causing an increase in the concentration of tyrosinate.

Figure 1 (right) of Reference 2 shows the fluorescence spectrum measured when the pressure of the egg white allergen protein was increased stepwise from 50 atm to 7,000 atm. As the pressure changed, the fluorescence intensity at a wavelength of 304 nm (derived from tyrosine) in the fluorescence spectrum decreased significantly, while at the same time the fluorescence intensity at a wavelength of 340 nm (derived from tyrosinate) increased. From this, it was assessed that a transition in tertiary structure occurred with the increase in pressure, causing a decrease in the proportion of structural states containing tyrosine and an increase in the proportion of structural states containing tyrosinate.

<CD and Fluorescence>
Reference 3 is an example of evaluating how the three-dimensional structure of lysozyme and the microenvironment of tryptophan residues in lysozyme change with temperature change based on CD and fluorescence spectra. In this method, an aqueous solution (0.05 mg/ml) of lysozyme (chicken egg white) is used as a sample, and the CD spectrum is measured in a 5 mm cell and the fluorescence spectrum is measured in a 10 mm cell at temperature intervals of 5°C from 20°C to 90°C.

Figure 1 in Reference 3 shows the results of measuring the temperature change of the CD spectrum in the wavelength range of 190 to 260 nm. The measurement conditions were a scan speed of 100 nm/min, a response of 2 sec, a bandwidth of 1 nm, a data interval of 0.1 nm, and two integrations. Figure 2 in Reference 3 shows the results of measuring the temperature change of the CD intensity at a wavelength of 220 nm.

Figure 4 in Reference 3 shows the results of temperature change measurements of the fluorescence spectrum in the wavelength range of 300 to 400 nm. The measurement conditions were an excitation wavelength of 280 nm, an excitation light bandwidth of 2 nm, a data interval of 1 nm, and a response of 1 sec. Figure 5 in Reference 3 shows the change in the ratio of the fluorescence intensity at a wavelength of 339 nm to that at a wavelength of 349 nm with a change in temperature.

According to the CD spectrum in Figure 1 of Reference 3, the intensity of the peak seen in the wavelength region below 200 nm decreases significantly with increasing temperature, and the negative peak at 207.7 nm at 20°C shifts to the shorter wavelength side to 202.8 nm at 90°C. Based on this, Reference 3 evaluates that the α-helix structure of lysozyme is dissolved by the increase in temperature, changing into a random coil structure. And, from the change in CD intensity at a wavelength of 220 nm resulting from the α-helix structure in Figure 2 of Reference 3, a rapid decrease is observed in the temperature range of 70 to 80°C, and the denaturation temperature is determined to be 73.9°C.

According to the fluorescence spectrum in Figure 4 of Reference 3, the fluorescence peak (absorption maximum) at 20°C, which was at 339 nm, shifts to longer wavelengths as the temperature rises, and appears at 348 nm at 90°C. In general, by monitoring the fluorescence of a protein, knowledge of the microenvironment in which the tryptophan residue is placed can be obtained. The fluorescence peak of the tryptophan residue reacts sensitively to the polarity of the surrounding environment. For example, when the tryptophan residue is buried inside the protein (non-polar environment), it shows a peak at around 330 nm. It is said that when the three-dimensional structure changes and the tryptophan residue comes into contact with water on the surface of the protein (highly polar environment), the peak shifts to around 350 nm. Therefore, Reference 3 evaluates that the change in the fluorescence spectrum in Figure 4 shows that the tryptophan residue buried inside the protein appears on the protein surface due to the thermal denaturation of lysozyme.

Furthermore, in Figure 5 of Reference 3, as in Figure 2, the peak ratio changes most significantly in the temperature range of 70 to 75°C, and therefore it is concluded that changes in the secondary structure (alpha helix structure) and tertiary structure (three-dimensional structure including tryptophan residues) of lysozyme occur in the same temperature range.

Suenaga, "Circular dichroism spectrometer (JASCO J-820)", page 7, [online], Kumamoto University Faculty of Pharmaceutical Sciences, Drug Discovery Research Center, Instrumental Analysis Facility, [Retrieved June 19, 2019], Internet <URL: http://iac.kuma-u.jp/equipment/details/pdf/23.pdf> Maeno, "Exploring hidden protein structures from high-pressure fluorescence experiments", Figure 1, [online], Biophysical Society of Japan, [Retrieved June 19, 2019], Internet <URL: https://www.biophys.jp/highschool/D-16.html> "Measurement of temperature change of lysozyme using J-820 + FMO-427L", Figure 1-5, [online], JASCO Corporation, [searched June 19, 2019], Internet <URL: https://www.jasco.co.jp/jpn/technique/200-CD-0005.pdf>

As shown in Patent Documents 1 to 3, conventional evaluation methods observe the respective spectra by CD measurement or fluorescence measurement, and evaluate whether or not the higher-order structure of the protein has changed by focusing on changes in the spectral shape in specific wavelength ranges. However, to evaluate the relative timing of different structural changes (such as changes in secondary and tertiary structures) occurring within the entire protein, or whether the entire protein has not undergone any structural changes, observing individual spectral data alone is insufficient, and judgment based on specialized knowledge and extensive experience is required.

The inventors believed that if there was a method for easily evaluating the relative timing of different structural changes occurring within the entire protein, or whether the entire protein is not undergoing any structural changes, it would be extremely useful for the development of industrial applications of proteins, including biopharmaceuticals.

The object of the present invention is to provide an evaluation method, an evaluation device, and an evaluation program that can easily evaluate multiple structural changes in a protein that occur when an external stimulus changes.

That is, the method for evaluating a structural change of a protein according to the present invention comprises the steps of:
A procedure for changing an external stimulus to a protein sample over time;
performing fluorescence spectrum measurement and CD spectrum measurement at a plurality of timings during a change in the external stimulus;
a step of calculating a simultaneous correlation spectrum and an anti-chronic correlation spectrum, which are two-dimensional correlation spectra for the time-dependent changes of the measured fluorescence spectrum and CD spectrum, with the measurement wavelength of the fluorescence spectrum as the X-axis and the measurement wavelength of the CD spectrum as the Y-axis;
displaying the simultaneous correlation spectrum and the cross-time correlation spectrum as image data on a display means;
and a step of evaluating the correlation between changes in the secondary structure and the tertiary structure of the protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum .

In the above-mentioned fluorescence spectrum measurement, the measurement wavelength range is set so as to include at least the wavelength range that responds to changes in the tertiary structure of the protein, and in the CD spectrum measurement, the measurement wavelength range is set so as to include at least the wavelength range that responds to changes in the secondary structure of the protein.

The external stimulus to the protein is preferably any one of temperature, pH, pressure, electric field, magnetic field, light, stress, protein concentration, type of buffer, and buffer concentration. It is also preferable to evaluate the correlation between the changes in the secondary structure and tertiary structure of the protein sample as shown in the following table.
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Simultaneous correlation Φ Cross-correlation Ψ Spectral changes at the cross peaks of λ1 and λ2
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Positive almost zero fluorescence and CD changes occur simultaneously in the same direction
The changes in fluorescence and CD are in the same direction, and fluorescence occurs before CD.
The positive and negative fluorescence and CD changes are in the same direction, and fluorescence occurs after CD.
Negative near-zero fluorescence and CD changes occur simultaneously in different directions
The changes in negative and positive fluorescence and CD are in opposite directions, and fluorescence occurs after CD.
The changes in fluorescence and CD are in opposite directions, and fluorescence occurs before CD .
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λ1: wavelength of the fluorescence spectrum that responds to changes in the tertiary structure of the protein
λ2: wavelength of the CD spectrum that responds to changes in the secondary structure of the protein

Furthermore, it is preferable to obtain the two-dimensional correlation spectrum for each of a plurality of protein samples as image data, and perform image principal component analysis on the image data. The plurality of protein samples can be grouped based on the principal components.

Next, the apparatus for evaluating a structural change in a protein according to the present invention comprises:
a receiving means for receiving, as data, fluorescence spectra and CD spectra measured at a plurality of timings during the change in an external stimulus applied to a protein sample over time;
a calculation means for calculating a simultaneous correlation spectrum and an intertemporal correlation spectrum, which are two-dimensional correlation spectra regarding the time-dependent changes in data of the fluorescence spectrum and the CD spectrum, with the measurement wavelength of the fluorescence spectrum being the X-axis and the measurement wavelength of the CD spectrum being the Y- axis;
a display command means for causing a display means to display the simultaneous correlation spectrum and the cross-time correlation spectrum as image data;
and an evaluation means for evaluating the correlation between changes in the secondary structure and the tertiary structure of the protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum .

The program for evaluating a structural change in a protein according to the present invention further comprises:
a receiving means for receiving, as data, fluorescence spectra and CD spectra measured at a plurality of timings during the change in an external stimulus applied to a protein sample over time;
a calculation means for calculating a simultaneous correlation spectrum and an intertemporal correlation spectrum, which are two-dimensional correlation spectra regarding the time-dependent changes of the measured fluorescence spectrum and the measured CD spectrum, with the measurement wavelength of the fluorescence spectrum being the X-axis and the measurement wavelength of the CD spectrum being the Y-axis ;
a display command means for causing a display means to display the simultaneous correlation spectrum and the cross-time correlation spectrum as image data;
The present invention is characterized in that the computer functions as an evaluation means for evaluating the correlation between changes in the secondary structure and tertiary structure of a protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum .

In evaluating protein stability (including evaluation of the manufacturing process), it can be said that more reliable evaluation is possible if the dynamic structural changes of the protein in response to changes in external stimuli over time, for example, the time lag in secondary and tertiary structural changes of the protein, can be properly understood. In the present invention, a two-dimensional correlation spectrum is calculated from the respective spectral data obtained by measuring the fluorescence spectrum and the CD spectrum of a protein accompanied by changes in external stimuli, and the two-dimensional correlation spectrum is displayed on a monitor, for example, as two-dimensional image data.

The calculation method for the two-dimensional correlation spectrum itself uses a known calculation method. In the present invention, the calculation method for the two-dimensional correlation spectrum is used to obtain a correlation between the change in fluorescence intensity at a certain wavelength (λ1) of the fluorescence spectrum and the change in CD intensity at a certain wavelength (λ2) of the CD spectrum. The above correlation is obtained for all or selected combinations of predetermined wavelengths (λ1, λ2) in a two-dimensional map. The two-dimensional correlation spectrum is a representation of the correlation results in a two-dimensional matrix with the wavelength of the fluorescence spectrum on the X-axis and the wavelength of the CD spectrum on the Y-axis. Usually, the two-dimensional correlation spectrum is calculated as a set of simultaneous correlation spectrum and different-time correlation spectrum.

By observing the image data of the two-dimensional correlation spectra (simultaneous correlation spectra and different-time correlation spectra), users can visually and comprehensively grasp simultaneous structural changes or multiple structural changes with a time lag that occur in a protein based on the positive or negative peaks that appear in the two-dimensional correlation spectra. In other words, it is now possible to grasp and evaluate complex structural changes in a protein at a glance. This type of evaluation method can contribute as an extremely useful analytical method in research, production, quality control, and other fields in the industrial use of proteins.

1 is a functional block diagram of a protein evaluation device according to a first embodiment. FIG. FIG. 2 is a diagram showing the overall configuration of a system including the evaluation device and a CD/fluorescence measuring device. FIG. 2 is a diagram showing an example of a sample unit of the CD/fluorescence measuring instrument. 1 is a flowchart showing a procedure for evaluating a structural change of a protein. 1A and 1B are diagrams showing examples of two-dimensional correlation spectra displayed on a display device, in which (A) shows a simultaneous correlation spectrum and (B) shows an intertemporal correlation spectrum. 11A and 11B are explanatory diagrams of a method for grouping multiple protein samples according to a second embodiment, in which (A) shows two-dimensional correlation spectrum images for multiple samples, and (B) shows the results of classifying the samples based on principal component analysis of the images.

1 is a functional block diagram of an evaluation device according to one embodiment of the present invention, which is used to evaluate structural changes in a protein. In this embodiment, the evaluation device 10 is composed of a computer, and the computer reads and executes an evaluation program stored in a storage medium, thereby functioning as, for example, a data receiving unit 12, a data holding unit 14, a correction unit 16, a calculation unit 18, a display command unit 20, and an evaluation unit 22.

The data receiving unit 12 receives fluorescence spectrum data and CD spectrum data of a protein sample from the outside. The data storage unit 14 is composed of a memory etc. and stores the spectrum data. The correction unit 16 corrects the spectrum data read from the data storage unit 14 so that the calculation of the two-dimensional correlation spectrum data is performed appropriately. The calculation unit 18 calculates the two-dimensional correlation spectrum data based on the fluorescence spectrum data and CD spectrum data. The display command unit 20 causes the data storage unit 14 to store the two-dimensional correlation spectrum data, and also causes the display device (such as a liquid crystal display) 24 to display the two-dimensional correlation spectrum data as an image. The display command unit 20 may also cause the display device 24 to display the fluorescence spectrum data and CD spectrum data as an image as appropriate.

The evaluation unit 22 displays evaluation information of the structural changes of the protein sample that appear in the two-dimensional correlation spectrum data on the display device 24 using characters or graphics.

On the display device 24, the evaluation information is displayed together with the two-dimensional correlation spectrum data, the fluorescence spectrum data, or the CD spectrum data. Alternatively, the evaluation information is displayed independently of these spectrum data. The display device 24 can employ a known means capable of displaying at least the two-dimensional correlation spectrum data as two-dimensional image data. Here, the display device 24 may be integrated with the evaluation device 10 or may be separate.

Figure 2 is an overall configuration diagram of a system consisting of the evaluation device 10 and the CD/fluorescence measuring device 30. The configuration of the CD/fluorescence measuring device 30 will be briefly explained using Figure 2. The CD/fluorescence measuring device 30 is a device that repeatedly measures CD spectrum data and fluorescence spectrum data at multiple timings during the change when an external stimulus is changed over time, using a protein sample as a measurement target. As shown in Figure 2, it is equipped with a light source lamp 31, a spectrometer 32, a polarization modulator (PEM) 33, a sample section 34, a photodetector for CD 35, a spectrometer for fluorescence 36, a photodetector for fluorescence 37, and a signal processing section 38 that processes signals from both photodetectors 35 and 37. The signal processing section 38 also sets the operating conditions of the measuring device and the measurement conditions. The signal processing section 38 executes drive control of the PEM 33, wavelength scanning control of each spectrometer 32 and 36, temperature control control of the sample section 34, cell replacement control, etc. according to the set conditions.

The irradiation optical system, which is composed of the light source lamp 31, the spectrometer 32, and the PEM 33 in that order, produces single-wavelength light whose polarization state changes periodically at the modulation frequency f of the PEM 33, and changes the selected wavelength of the spectrometer 32 within a predetermined wavelength range. In this embodiment, for each selected wavelength, circularly polarized light that alternates between left and right at a period determined by the modulation frequency of the PEM 33 is produced, and the sample section 34 is irradiated with the light.

Figure 3 is a plan view of the temperature-controlled cell holder 39 and sample cell 40 arranged in the sample section 34. The temperature-controlled cell holder 39 is, for example, a water-cooled cell holder with a built-in temperature control element such as a Peltier element, and can adjust the temperature of the sample cell 40 in the cell holder and change the temperature over time. The sample cell 40 is, for example, a micro quartz cell with an internal space having a rectangular cross section. Depending on the state of the protein sample (usually a mixture of a medium such as a liquid and a protein), the sample cell 40 is appropriately selected from a configuration that holds the protein sample inside the cell and a configuration that continuously flows the protein sample.

The single-wavelength light from the PEM 33 enters through one of the window plates of the sample cell 40, passes through the protein sample inside, exits through the other window plate, and is then detected by the CD photodetector 35.

<Measurement of CD spectrum>
When single-wavelength light with alternating left-right circular polarization passes through a circular dichroism site contained in a protein sample, a difference occurs between the absorbance of the left-handed circularly polarized light and the absorbance of the right-handed circularly polarized light that passes through the protein sample. This difference appears as an AC component in the light intensity signal from the photodetector 35. The signal processor 38 detects the AC component from the light intensity signal using a reference signal with the modulation frequency f of the PEM 3, and obtains a CD intensity signal. In addition, when the spectrometer 32 is driven to scan the wavelength of the single-wavelength light, a CD intensity signal corresponding to the wavelength is obtained, and the signal processor 38 can obtain CD spectrum data. FIG. 3 shows a schematic diagram of the CD spectrum data of a protein sample.

The sample cell 40 further has a third window plate that is perpendicular to the pair of windows described above.

<Measurement of fluorescence spectrum>
2, in the fluorescence measurement, the spectrometer 32 at the front end of the sample section fixes the wavelength of the excitation light, and the spectrometer 36 at the rear end scans the wavelength of the fluorescence from the protein sample, thereby making it possible to measure fluorescence spectrum data (constant excitation light wavelength). Also, the spectrometer 32 at the front end scans the wavelength of the excitation light, and the spectrometer 36 at the rear end selects a component of a constant wavelength from the fluorescence emitted from the protein sample, thereby making it possible to measure fluorescence spectrum data (constant fluorescence wavelength).

Either type of fluorescence spectrum data may be used. Here, the former case will be described. When the wavelength of the single-wavelength light (excitation light) from the PEM 33 is constant, for example 280 nm, and the single-wavelength light excites a fluorescent site (for example, a fluorescent probe) contained in a protein sample, the protein sample emits fluorescence. The fluorescence is emitted from the third window plate of the sample cell 40, enters the fluorescence spectrometer 36, where it is dispersed into fluorescence of each wavelength, and is then detected by the photodetector 37. By driving the fluorescence spectrometer 36 to scan the selected wavelength, the signal processing unit 38 can obtain a fluorescence intensity signal for each selected wavelength from the photodetector 37 and acquire fluorescence spectrum data. Figure 3 shows a schematic diagram of the fluorescence spectrum data of a protein sample.

During fluorescence spectrum measurement, the PEM33 may be kept modulated or the modulation may be stopped.

Conventionally, when measuring spectra separately using independent CD and spectrofluorometers, or when measuring spectra using a single CD measurement instrument with a CD cell and a fluorescence cell separately placed in the sample section, it was extremely difficult to synchronize the changes over time in external stimuli (especially temperature) of the protein sample in the two sample cells.

In this embodiment, by adopting the sample section 34 configured as shown in FIG. 3, both the CD spectrum and the fluorescence spectrum are measured from a protein sample in the same sample cell. In other words, each spectrum can be measured simultaneously using one sample cell 40, eliminating the "deviation" caused by external stimuli that occurs when measuring with two sample cells. In addition, since two sets of spectral data can be obtained in one measurement, the amount of sample required for measurement can be reduced.

The flowchart in Figure 4 shows the procedure (S1 to S8) for evaluating protein structural changes using the system configuration in Figure 2.

In step S1, the operating conditions of the CD/fluorescence measuring device 30 are set. For example, in fluorescence measurement, the measurement wavelength range is set so that it includes at least the wavelength range that responds to changes in the tertiary structure of proteins. In CD measurement, the measurement wavelength range is set so that it includes at least the wavelength range that responds to changes in the secondary structure of proteins.

In step S2, the conditions for the time-dependent change of the external stimulus to the protein sample are set. The external stimulus can be selected from physical parameters such as the protein temperature, pH, pressure, electric field, magnetic field, light, stress, protein concentration, buffer type, and buffer concentration. In this embodiment, as an example, the temperature-controlled cell holder 39 is used to set the conditions for changing the temperature of the protein sample in the sample cell 40 from 20°C to 90°C over time.

In step S3, the temperature-controlled cell holder 39 is driven under the above conditions to generate a change in the external stimulus over time. Then, while changing the temperature of the protein sample over time from 20°C to 90°C, both the fluorescence spectrum and the CD spectrum are repeatedly measured at temperature intervals of, for example, 5°C. At each temperature interval of 5°C, the signal processing unit 38 processes the intensity signals from each of the photodetectors 35, 37 to obtain CD spectrum data and fluorescence spectrum data, and outputs these data to the evaluation device 10. As a result of performing steps up to S3, the fluorescence spectrum and CD spectrum are simultaneously obtained at each temperature of 20°C, 25°C, ..., 90°C.

Steps S4 to S8 show the flow of spectral data processing by the computer (evaluation device 10), and processing may be started continuously after step S3 is completed. Alternatively, the CD spectral data measured by a different CD measuring device and the fluorescence spectral data measured by a different spectrofluorometer may be stored in the data storage unit 14 with the time-dependent change of the external stimulus as a common condition, and the computer may execute an evaluation program for these spectral data as required by the user.

Here, steps S4 to S8 will be described as a series of operations following step S3. First, the computer receives each piece of spectral data from the signal processing unit 38 and stores it in the data storage unit 14, such as a memory (step S4). Here, the computer can make advance corrections to each piece of spectral data read from the data storage unit 14 in order to properly calculate the two-dimensional correlation spectral data (step S5).

<Spectral data correction details>
For the fluorescence spectrum data, a high-order light cut process for the excitation light, dark correction, and spectrum correction are appropriately performed, and for the CD spectrum data, baseline correction and concentration correction during titration measurement are appropriately performed.

Next, the computer calculates two-dimensional correlation spectrum data for the changes in the fluorescence spectrum data and the changes in the CD spectrum data, with the measurement wavelength of the fluorescence spectrum data on the X-axis and the measurement wavelength of the CD spectrum on the Y-axis (step S6).

<Calculation of two-dimensional correlation spectrum>
Two-dimensional correlation spectroscopy is known as a method for analyzing time-dependent changes in a spectrum (also called "dynamic spectrum"). If you simply need to calculate a two-dimensional correlation spectrum from general dynamic spectrum data, you can use commercially available software.

Here, the wavelength of the spectral data is λ, the dynamic fluorescence spectrum is represented by (1), and the dynamic CD spectrum is represented by (2).

If the dynamic fluorescence spectrum (3) and the dynamic CD spectrum (4) are measured at arbitrary wavelengths λ1 and λ2 with a certain correlation time τ, the cross-correlation function "X(τ)" of the following equation (5) can be obtained.

This correlation function "X(τ)" can also be expressed using a simultaneous correlation intensity Φ, which corresponds to the real part when the correlation function is expressed as a complex number, and an intertemporal correlation intensity Ψ, which corresponds to the imaginary part. The simultaneous correlation intensity Φ and the intertemporal correlation intensity Ψ are two-dimensional spectra determined by two independent wavelengths λ1 and λ2.

The computer calculates the simultaneous correlation spectrum and the different time correlation spectrum, which are the most basic two-dimensional correlation spectra (step S6), and displays the calculated two-dimensional correlation spectra on a display means such as an LCD display (step S7).

Figures 5 (A) and (B) are schematic diagrams showing examples of a simultaneous correlation spectrum Φ(λ1, λ2) and an anti-chronic correlation spectrum Ψ(λ1, λ2) displayed on a monitor or the like. The peaks of each correlation spectrum are represented by changing colors according to their height, but they may also be displayed as contour lines or in a three-dimensional display.

Finally, the computer evaluates changes in the secondary and tertiary structures of the protein sample based on the positive or negative cross peaks that appear in the two-dimensional correlation spectrum (step S9).

Here, we briefly explain how to evaluate structural changes in protein samples.

In the simultaneous correlation spectrum Φ(λ1, λ2) in FIG. 5(A), a positive cross peak P11 occurs at the intersection of the fluorescence wavelength λ11 (near 330 nm) and the CD wavelength λ21 (near 200 nm). In other words, the intensities of the fluorescence wavelength λ11 and the CD wavelength λ21 are simultaneously correlated in the positive direction.

Positive simultaneous correlation (cross peak P11) occurs when both the fluorescence signal and the CD signal decrease (or increase), and it can be said that the direction of change in the respective spectral intensities is the same. Fluorescence data at around 330 nm indicates a tryptophan-induced tertiary structure change, while CD data at 200-230 nm indicates an α-helix secondary structure change. In this example, this can be evidence that both the protein's secondary structure (e.g., the α-helix structure) and tertiary structure (e.g., a three-dimensional structure containing a fluorescent probe) have collapsed, or that both secondary and tertiary structures have been formed.

When a negative cross peak appears in the simultaneous correlation spectrum and the intensities of the fluorescence spectrum and CD spectrum are simultaneously correlated in the negative direction (also called anti-correlation), one of the fluorescence signal and the CD signal decreases while the other increases. This can be evidence that, for example, there is no change in one of the secondary and tertiary structures of a protein, but a change such as collapse or formation has occurred in the other.

Thus, the presence of cross peaks in the simultaneous correlation spectrum is significant as it indicates the possible interaction between changes in the secondary and tertiary structures of a protein in response to time-dependent changes in external stimuli.

On the other hand, the anisochronic correlation spectrum Ψ(λ1, λ2) in FIG. 5(B) also has a positive cross peak P21 at the intersection of the fluorescence wavelength λ12 (near 330 nm) and the CD wavelength λ21 (near 200 nm). In other words, the intensities of the fluorescence wavelength λ12 and the CD wavelength λ21 are anisochronically correlated in the positive direction.

As shown in Figures 5 (A) and (B), when a positive synchronous correlation (cross peak P11) and a positive asymmetric correlation (cross peak P21) occur for a certain wavelength combination (λ1, λ2), the directionality of the changes in fluorescence and CD is the same, with the change in fluorescence intensity occurring first and the change in CD intensity occurring later. For example, this could be evidence that the change in the tertiary structure of a protein proceeds first, followed by the start of a change in the secondary structure.

When positive simultaneous correlation and negative asymmetric correlation occur, the directionality of the changes in fluorescence and CD is the same, but the change in CD intensity occurs first, followed by the change in fluorescence intensity. For example, this could be the basis for the fact that changes in the secondary structure of a protein occur first, followed by changes in the tertiary structure.

In other words, in response to a time-dependent change in an external stimulus to a protein, a change in the fluorescence intensity around 330 nm (a change in the tertiary structure) occurs first, followed by a change in the CD intensity around 200 nm (a change in the secondary structure); this allows us to grasp the time lag between these structural changes. It is now possible to determine the order in which the secondary and tertiary structures of a protein change in response to a time-dependent change in an external stimulus, making it easier to evaluate the thermal stability of a protein.

Furthermore, combinations of wavelengths that do not show cross peaks in the cross-chronological correlation spectrum indicate that the changes in signal intensity are synchronized, meaning that the speed of structural changes is the same, or that no structural changes are occurring in both.

Examples of the display of the evaluation contents by the computer's evaluation unit 72 are shown in Figures 5 (A) and (B). Alternatively, the display may be in a table format or in text.

The following table shows an example of how to read a two-dimensional correlation spectrum (Φ, Ψ). λ1 indicates the wavelength of the fluorescence spectrum, and λ2 indicates the wavelength of the CD spectrum. Based on Table 1, by reading the two-dimensional correlation spectrum image in Figure 5, it can be seen that under the same temperature change conditions, the protein sample experienced a tryptophan-derived tertiary structure change (change in fluorescence) before a secondary structure change in the α-helix (change in CD). The evaluation results based on Table 1 may be displayed on the two-dimensional correlation spectrum image in Figure 5.

[Table 1]
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Simultaneous correlation Φ Antichronic correlation Ψ Spectral changes at the cross peaks of λ1 and λ2 ------------------------------------------------
Positive Almost zero The changes in fluorescence and CD occur simultaneously in the same direction. Positive Positive The changes in fluorescence and CD occur in the same direction, with fluorescence occurring before CD. Positive Negative The changes in fluorescence and CD occur in the same direction, with fluorescence occurring after CD. Negative Almost zero The changes in fluorescence and CD occur simultaneously in different directions. Negative Positive The changes in fluorescence and CD occur in different directions, with fluorescence occurring after CD. Negative Negative The changes in fluorescence and CD occur in different directions, with fluorescence occurring before CD.------------------------------------------------

In fields such as antibody drug development, properly understanding temperature-dependent structural changes in proteins is an important issue in product development, establishment of manufacturing processes, and quality control. In this embodiment, changes in the secondary and tertiary structures of proteins are monitored, making it easy to evaluate structural changes in proteins, and therefore important information for confirming the effects of functional sites imparted to proteins and determining the presence or absence of mutations can be easily obtained. This is expected to be widely used in the field of antibody drugs, where target sites are designed these days.

Second embodiment Fig. 6 is a diagram showing that a computer performs principal component analysis for images using two-dimensional correlation spectrum data of a plurality of protein samples as images. In this embodiment, the system of the first embodiment (Fig. 2) is used, and a six-stage temperature-controlled cell changer (with built-in water-cooled Peltier element) is used as the temperature-controlled cell holder 39 in Fig. 3, so that spectrum measurements of six samples can be performed simultaneously or sequentially. In addition, the evaluation device 10 performs principal component analysis for images.

For example, we will explain a case in which, during the development stage of a biopharmaceutical, one sample consisting of a native protein and five samples to which some functional group has been added under different conditions are used to confirm whether or not there is any effect due to the addition of a functional group to the protein.

First, under temperature change conditions, the system of this embodiment is used to acquire the fluorescence spectrum data and CD spectrum data of these six samples, and six sets of two-dimensional correlation spectrum data are obtained (FIG. 6(A)). Then, these six sets of two-dimensional correlation spectra (synchronous correlation spectrum, different-time correlation spectrum) are used as image data to perform principal component analysis of these images. For this image analysis, commercially available principal component analysis software for images can be used.

As a result of the principal component analysis of the images, the six samples can be divided into groups. FIG. 6(B) shows an example using the first principal component (PC1) of the image data and the second principal component (PC2) of the image data. For example, the six samples can be grouped based on the results of the match/mismatch between the metachronic correlation spectral image of a native protein and the metachronic correlation spectral image of another sample. Samples that belong to the same group in the metachronic correlation spectral image can also be further divided into finer groups based on the results of the principal component analysis of the simultaneous correlation spectral image.

In the example of Figure 6 (B), group 1 contains multiple samples, including a native sample (No. 1). These samples are highly consistent with the native sample and can be used as a basis for determining that no structural changes have occurred in the protein.

In the case of temperature change conditions, using a sample cell like that shown in Figure 3 to simultaneously perform two types of spectrum measurements has the advantage that the temperature conditions of the protein sample can be matched when performing the two types of spectrum measurements. However, even if fluorescence measurement and CD measurement are performed separately, simultaneous measurement of two types of spectra is not essential as long as it is possible to match the temperature changes of each protein sample.

The evaluation method of the present invention can also be applied when measuring two types of spectra using a CD measuring device and a fluorescence measuring device.

10 Evaluation device (computer)
12 Data receiving unit (receiving means)
18 Calculation unit (means for calculating two-dimensional correlation spectrum)
20 Display command unit (display command means)
22 Evaluation unit (evaluation means for evaluating structural changes in a protein sample)
24 Display device (display means)
30 CD/fluorescence measuring device 34 Sample section 38 Signal processing section 39 Temperature-controlled cell holder (6-way temperature-controlled cell changer)
40 Sample cell

Claims (6)

A procedure for changing an external stimulus to a protein sample over time;
performing fluorescence spectrum measurement and CD spectrum measurement at a plurality of timings during a change in the external stimulus;
a step of calculating a simultaneous correlation spectrum and an anti-chronic correlation spectrum, which are two-dimensional correlation spectra for the time-dependent changes of the measured fluorescence spectrum and CD spectrum, with the measurement wavelength of the fluorescence spectrum as the X-axis and the measurement wavelength of the CD spectrum as the Y-axis;
displaying the simultaneous correlation spectrum and the cross-time correlation spectrum as image data on a display means;
evaluating correlations of changes in the secondary and tertiary structures of the protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum ;
A method for evaluating a structural change of a protein, comprising: The method for evaluating a structural change of a protein according to claim 1,
The method for evaluating a structural change of a protein, wherein the external stimulus is any one of temperature, pH, pressure, an electric field, a magnetic field, light, stress, protein concentration, a type of buffer, and a buffer concentration. 3. The method for evaluating a structural change in a protein according to claim 1, wherein the correlation between the changes in the secondary structure and the tertiary structure of the protein sample is evaluated as shown in Table 1.
[Table 1]
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Simultaneous correlation Φ Cross-correlation Ψ Spectral changes at the cross peaks of λ1 and λ2
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Positive almost zero fluorescence and CD changes occur simultaneously in the same direction
The changes in fluorescence and CD are in the same direction, and fluorescence occurs before CD.
The positive and negative fluorescence and CD changes are in the same direction, and fluorescence occurs after CD.
Negative near-zero fluorescence and CD changes occur simultaneously in different directions
The changes in negative and positive fluorescence and CD are in opposite directions, and fluorescence occurs after CD.
The changes in fluorescence and CD are in opposite directions, and fluorescence occurs before CD .
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λ1: wavelength of the fluorescence spectrum that responds to changes in the tertiary structure of the protein
λ2: wavelength of the CD spectrum that responds to changes in the secondary structure of the protein The method for evaluating a structural change of a protein according to any one of claims 1 to 3 ,
A method for evaluating structural changes in a protein, comprising obtaining the two-dimensional correlation spectrum as image data for each of a plurality of protein samples, and performing image principal component analysis on the image data. a receiving means for receiving, as data, fluorescence spectra and CD spectra measured at a plurality of timings during the change in an external stimulus applied to a protein sample over time;
a calculation means for calculating a simultaneous correlation spectrum and an intertemporal correlation spectrum, which are two-dimensional correlation spectra regarding the time-dependent changes in data of the fluorescence spectrum and the CD spectrum, with the measurement wavelength of the fluorescence spectrum being the X-axis and the measurement wavelength of the CD spectrum being the Y- axis;
a display command means for causing a display means to display the simultaneous correlation spectrum and the cross-time correlation spectrum as image data;
an evaluation means for evaluating the correlation between changes in the secondary structure and the tertiary structure of the protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum ;
An apparatus for evaluating a structural change in a protein, comprising: a receiving means for receiving, as data, fluorescence spectra and CD spectra measured at a plurality of timings during the change in an external stimulus applied to a protein sample over time;
a calculation means for calculating a simultaneous correlation spectrum and an intertemporal correlation spectrum, which are two-dimensional correlation spectra regarding the time-dependent changes of the measured fluorescence spectrum and the measured CD spectrum, with the measurement wavelength of the fluorescence spectrum being the X-axis and the measurement wavelength of the CD spectrum being the Y-axis ;
a display command means for causing a display means to display the simultaneous correlation spectrum and the cross-time correlation spectrum as image data;
A program for evaluating structural changes in proteins, which causes a computer to function as an evaluation means for evaluating the correlation between changes in the secondary structure and tertiary structure of a protein sample based on positive or negative peaks appearing in the simultaneous correlation spectrum and the different time correlation spectrum.

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