A method for analyzing the structure and dynamic behavior in solution of kappa-carrageenase

Abstract

The application provides a method for analyzing the structure and dynamic behavior of kappa-carrageenase in a solution, which utilizes the HolE to analyze the changes of the substrate channel of wild-type kappa-carrageenase and mutant N205C-G239C; utilizes the JEM-1400 Flash transmission electron microscope to observe the reaction of wild-type kappa-carrageenase and mutant N205C-G239C in an environment containing a substrate kappa-neo-carrageenan tetrasaccharide under an electron energy of 100 keV and a dose rate; wherein the sample is photographed with an exposure time of 0.1 seconds, and the image is processed and analyzed by using ImageJ. The method utilizes the mutant with higher stability and designs the observation system and conditions, can reveal the structure and function relationship of the protein, research the dynamic behavior of the protein, and provides a theoretical basis for the application of liquid phase electron microscopy in the field of protein observation.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bioengineering, and particularly relates to a method for analyzing the structure and dynamic behavior in solution of kappa-carrageenase. BACKGROUND

[0002] In recent years, with the development of protein engineering technology, wild-type carrageenase is subjected to molecular modification in order to improve the thermal stability, enzyme activity and other enzymatic properties of carrageenase, expand the application range of carrageenase, and promote the high-value utilization of carrageenase.

[0003] The structure of a protein often determines its function. Common electron microscopes play an important role in structure analysis, but cannot meet the demand for analyzing conformational changes of molecules over time. Traditional electron microscopes usually only need to be observed under vacuum or dry conditions, and therefore cannot observe the dynamic changes of molecules. The main difference between liquid-phase electron microscopy and other electron microscopes for observing structures is that it can observe samples in a liquid environment, which also enables liquid-phase electron microscopy to better simulate the real environment in vivo and observe the structure and dynamic behavior of biological molecules in solution. In addition, liquid-phase electron microscopy can also achieve in-situ observation, that is, when a sample is subjected to external stimulation or changes in conditions, it can also observe the response and changes of the sample in real time. In general, liquid-phase electron microscopy is closer to the real environment in vivo when observing samples, and can provide more information about the structure and behavior of biological molecules.

[0004] Liquid-phase electron microscopy has higher requirements for sample stability, resolution and contrast during testing. First, liquid-phase electron microscopy needs to observe samples in a liquid environment, which puts higher requirements on the stability of samples, sample preparation and operation, etc. Proteins may be damaged or inactivated in liquid-phase electron microscopy because proteins are more sensitive to temperature, pH and other conditions. Furthermore, the resolution and contrast of liquid-phase electron microscopy are usually low, which may not be fine enough for observing the fine structure of biological molecules, especially in the biological field, higher resolution electron microscopes are usually needed to observe the biological structure of proteins. Finally, in liquid-phase electron microscopy, sample preparation and operation are relatively complex, requiring special techniques and equipment. For biological samples, especially living biological samples, sample handling and operation are more difficult, which may lead to inactivation or denaturation of the sample.

[0005] Therefore, the way of analyzing the structure and dynamic behavior in solution of kappa-carrageenase needs to be improved. SUMMARY

[0006] This invention aims to at least partially solve one of the technical problems in the aforementioned technologies. To this end, this invention proposes a method for analyzing the structure and dynamic behavior of κ-carrageenanase in solution. This method utilizes a more stable mutant and designs an observation system and conditions that can reveal the structure-function relationship of proteins and study their dynamic behavior, providing a theoretical basis for the application of liquid electron microscopy in the field of protein observation.

[0007] Therefore, in embodiments of the present invention, a method for analyzing the structure and dynamic behavior of κ-carrageenase in solution is provided, comprising the following steps:

[0008] S1. Hole analysis was used to analyze the changes in substrate channels of wild-type κ-carrageenase and mutant N205C-G239C.

[0009] S2. Using a JEM-1400 Flash transmission electron microscope at an electron energy of 100 keV and At a dose rate of [specific value], the reactions of wild-type κ-carrageenase and mutant N205C-G239C in an environment containing the substrate κ-neocarrageenone were observed; samples were photographed with an exposure time of 0.1 seconds, and the images were processed and analyzed using ImageJ.

[0010] According to an embodiment of the present invention, by using Hole analysis to analyze the changes in substrate channels of wild-type κ-carrageenase and the mutant N205C-G239C, the size of the protein substrate channels can be obtained, proving that the mutation enlarges the protein substrate channels; using a JEM-1400 Flash transmission electron microscope at an electron energy of 100 keV and By observing at specific dose rates, the process of protein catalytic tunnel opening can be observed. Furthermore, by using mutants with improved stability as suitable observation materials, it is ensured that the protein will not be inactivated during the observation process. This allows for the analysis of the structure and dynamic behavior of κ-carrageenanase in solution. Under the premise of achieving in-situ observation, it not only helps to reveal the structure-function relationship of proteins and study their dynamic behavior, but also expands the application field of liquid phase electron microscopy by successfully observing protein movement, so as to facilitate the rational modification of specific target enzymes.

[0011] Optionally, step S2 includes:

[0012] Mix κ-carrageenase with κ-neocarboxylic acid substrate to prepare a liquid protein for testing;

[0013] The protein liquid to be tested was dropped onto a gold grid containing a graphene support film, etched, and then inverted into a beaker containing HPLC-grade water to prepare a microchip sample.

[0014] The microchip sample is placed in a JEM-1400 Flash transmission electron microscope for observation.

[0015] Optionally, the amino acid sequence of the mutant N205C-G239C is shown as SEQ ID NO. 1.

[0016] Optionally, in step S1, the minimum and maximum diameters of the substrate channel of the wild-type kappa-carrageenase are 0.73 nm and 0.90 nm respectively, and the minimum and maximum diameters of the substrate channel of the mutant N205C-G239C are 0.75 nm and 1.29 nm respectively.

[0017] Optionally, in step S2, the process of opening of the protein catalytic tunnel is observed.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Experimental results for protein thermostability determination according to embodiments of the present application;

[0020] Figure 2 Analysis results of substrate channel diameter according to embodiments of the present application (green color for WT, and purple color for mutant N205C-G239C);

[0021] Figure 3 Results of dynamic change process of protein substrate channel observed by liquid phase electron microscopy according to embodiments of the present application, wherein, TEM original image (A) and image after adjusting appropriate contrast (B) and corresponding schematic diagram (C) of WT liquid-air interface movement, TEM original image (D) and image after adjusting appropriate contrast (E) and corresponding schematic diagram (F) of N205C-G239C liquid-air interface movement. DETAILED DESCRIPTION

[0022] Embodiments of the present application are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference signs represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by reference to the accompanying drawings are exemplary and are intended to explain the present application, and cannot be understood as limiting the present application.

[0023] The following disclosure provides many different embodiments or examples for implementing different implementations of the application. For simplicity of the disclosure, the following description provides specific examples of the application. Needless to say, they are merely examples and are not intended to limit the application. Moreover, the examples of the various specific processes and materials provided by the application are intended to be illustrative only and are not intended to limit the scope of the application. Unless otherwise specified, the practice of the application will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, and the like, which are within the capabilities of a person of ordinary skill in the art. In addition, unless otherwise specified, nucleic acids are written left to right in 5' to 3' orientation and amino acid sequences are written left to right in amino to carboxy orientation.

[0024] The application will now be described by way of illustrative specific examples, which are not intended to limit the scope of the application in any way. It is specifically noted that the reagents used in the application are commercially available unless otherwise specified.

[0025] Example 1 Protein thermostability assay

[0026] Thermostability: 10 μL enzyme solution (10 ng / μL) was incubated at different temperatures (40, 45, 50, 55, 60 °C) for 30 min, placed on ice for 5 min, and then 490 μL of 0.5% (w / v) carrageenan substrate solution (0.5% (w / v) in 50 mmol / L Na2HPO4-NaH2PO4 buffer (pH 8.0)) was added. Both enzymes were reacted at 50 °C for 10 min, inactivated by boiling for 5 min, and then 500 μL of DNS reagent was added. After boiling for 10 min and cooling, the absorbance was measured at 520 nm. The relative enzyme activity was determined based on the enzyme activity of the enzyme without incubation as 100%. The enzyme solution was wild-type kappa-carrageenase (Genebank ID GU386342) and enzyme mutant N205C-G239C (Chinese invention patent application CN 115725548 A).

[0027] The results, as shown in Figure 1 Table 1, mutant N205C-G239C has stronger thermostability and is more suitable as a protein sample to be tested.

[0028] Example 2 Protein substrate channel analysis

[0029] Pseudoalteromonas porphyrae LL1 kappa-carrageenase (GenBank No. GU386342) was modeled using AlphaFold2 (https: / / github.com / sokrypton / ColabFold) and the model was validated using the SAVES v6.0 suite of tools (https: / / saves.mbi.ucla.edu / ). HOLE (http: / / www.holeprogram.org / ) was used to evaluate the substrate channel in the kappa-carrageenase trajectory.

[0030] The results, as shown in Figure 2 The minimum and maximum diameters of the WT substrate channel were 0.73 nm and 0.90 nm, respectively, and the minimum and maximum diameters of the substrate channel of the mutant N205C-G239C were 0.75 nm and 1.29 nm, respectively, so that the mutation made the substrate channel of the carrageenase larger to facilitate subsequent liquid-phase electron microscopy observation.

[0031] Example 3 Liquid-phase electron microscopy observation of dynamic changes in proteins

[0032] Preparation of the sample to be tested: The substrate kappa-neo-carrageenan tetrasaccharide (purchased from Shandong Qingdao Bozhihuilier Biotechnology Co., Ltd.) was dissolved in H2O to 0.05 μM. The kappa-carrageenase WT and mutant N205C-G239C were diluted with PBS solution (50 mM NaH2PO4-Na2HPO4, pH 8.0) to 0.05 μM. 50 μL of kappa-carrageenase sample was mixed with 50 μL of kappa-neo-carrageenan tetrasaccharide substrate.

[0033] Preparation of the microchip sample (covered with graphene): In order to maintain the environment required for the physiology of the protein, it is necessary to cover the liquid to be tested (protein and substrate) with a gold carrier net containing graphene (purchased from ACS Material LLC). Operation steps: The test was carried out at room temperature, and the protein liquid to be tested (50 μL) was dropped on the gold carrier net containing the graphene support film. After etching to remove the copper protective layer provided by the manufacturer, it was quickly inverted in a beaker containing HPLC-grade water (containing a floating graphene layer), and a pair of fine tweezers was used to slowly pick up the microchip sample formed. Photography and data analysis: The detection was carried out using a JEM-1400 Flash transmission electron microscope (purchased from JEOL) at an electron energy of 100 keV and a magnification of 100,000 times. The results are shown in FIG. 1. were performed at a dose rate of 0.1 e- / A2 / s. The samples were photographed with an exposure time of 0.1 s. The images were processed and analyzed using ImageJ. The dynamic process of the protein catalytic tunnel was observed by liquid phase electron microscopy (LP-EM) for the reaction of WT and N205C-G239C in the environment containing the substrate kappa-neocallimastix tetrasaccharide, and the dynamic process of the protein catalytic tunnel was indeed observed, and the protein was constantly moving in the liquid environment (proving that the photographed protein was active) (videos 1 and 2); in Figure 3 In (E), the process of opening of the protein catalytic tunnel was observed, starting from the conformational closed state (0 s) and finally gradually returning to the closed conformation (8 s). In Figure 3 In (E), the process of opening of the protein catalytic tunnel was observed, starting from the conformational closed state (0 s) and finally gradually returning to the closed conformation (8 s). In

[0034] In summary, according to the embodiments of the present application, the mutant N205C-G239C with higher stability is used, and the observation system and conditions are designed, and the system suitable for the observation of the protein and the stable observation conditions are successfully researched, which not only helps to reveal the structure and function relationship of the protein, study the dynamic behavior of the protein, and successfully observe the movement of the protein, but also expands the application field of liquid phase electron microscopy, so as to facilitate the rational modification of the enzyme for a specific purpose.

[0035] In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" and the like means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the present specification, the illustrative description of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in the present specification.

[0036] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above embodiments within the scope of the present application.

Claims

1. A method of analysing the structure and dynamic behaviour in solution of a kappa-carrageenase, characterised in that, The method comprises the following steps: S1, analyzing the change of substrate channel of wild type kappa-carrageenase and mutant N205C-G239C by Hole; S2, mixing kappa-carrageenase and kappa-neo carrageenan tetrasaccharide substrate into a protein liquid to be tested; The protein liquid to be tested is dropped on a gold carrier net containing a graphene support film, etched, inverted in a beaker containing HPLC grade water, and a microchip sample is prepared; The microchip sample is placed in a JEM-1400Flash transmission electron microscope for observation, and the reaction of wild type kappa-carrageenase and mutant N205C-G239C in an environment containing substrate kappa-neo carrageenan tetrasaccharide is observed by JEM-1400Flash transmission electron microscope at an electron energy of 100 keV and a dose rate of 10-20 e- / (Å2 s); wherein the sample is photographed with an exposure time of 0.1 second, and the image is processed and analyzed using ImageJ; The amino acid of the mutant N205C-G239C is the sequence shown in SEQ ID NO.

1.

2. The method of claim 1, wherein, In step S1, the minimum and maximum diameters of the substrate channel of wild type kappa-carrageenase are 0.73 nm and 0.90 nm respectively, and the minimum and maximum diameters of the substrate channel of mutant N205C-G239C are 0.75 nm and 1.29 nm respectively.

3. The method of claim 1, wherein, In step S2, the process of opening the protein catalytic tunnel is observed.

Images