CN106932375A - The bio-orthogonal Raman in-situ detection method of protein conformation change - Google Patents

Abstract

The present invention relates to a bio-orthogonal Raman in situ detection method for protein conformation changes, in particular to a method for using a bio-orthogonal Raman reporter group to specifically label and detect protein conformation changes, which comprises the following steps: a) preparing a carrier The non-natural amino acid of the bio-orthogonal Raman reporter group; b) using the gene extension technology to insert the non-natural amino acid prepared in step a) into the target protein, so as to realize the bio-orthogonal Raman reporter group labeling of the target protein; c ) using bio-orthogonal Raman technology to detect the protein obtained in step b). The method of the present invention uses gene codon amplification technology and SERS enhanced method to sensitively and efficiently detect the conformational changes of proteins with bio-orthogonal groups under different environmental conditions or activity conditions, and realizes the analysis of solutions and cell systems The link between protein conformation and function in .

Description

Bioorthogonal Raman in situ detection of protein conformational changes

technical field

The present invention relates to the detection of protein chemical and conformational changes, in particular to a method of using bio-orthogonal Raman reporter group to specifically label and detect proteins, using bio-orthogonal Raman technology to detect protein conformation changes in vitro, and using bio-orthogonal Raman Mann technology is a method for in situ detection of protein conformational changes in living cells.

Background technique

The spatial conformation of a protein molecule is the basis of its function and activity. The response of proteins to the environment and stimuli is through the conformational changes of proteins or protein complexes to realize the regulation of their functions and activities. The positions, orientations, and movements of some important amino acids undergo significant changes during the protein's allosteric process. Therefore, rapid, sensitive and efficient detection of protein conformational changes (especially in situ detection of protein conformational changes under physiological conditions) is crucial for the study of protein structure-function linkages.

At present, there are many methods for studying protein structure, including X-ray crystallography, nuclear magnetic resonance, infrared spectroscopy, fluorescence spectroscopy, Raman spectroscopy, circular dichroism spectroscopy, mass spectrometry, etc. Each of these methods has advantages and disadvantages and scope of application. For example, X-ray crystallography has been developed for decades. The sample does not need to be labeled, the molecular weight is not limited, and the data collection and analysis are relatively mature. It can provide protein structures at atomic resolution; but X-ray crystallography requires the analysis of protein Crystals (preparing protein single crystals are very complicated and difficult), and require a large sample size, time-consuming, poor sensitivity, suitable for steady-state proteins that are easy to obtain crystals, and difficult to apply to transient conformational changes in solution. Infrared spectroscopy has low sensitivity (the content required in component analysis should generally be greater than 0.1%), and is severely interfered by the signal of water. The near-infrared technology developed later failed to solve this problem, making infrared spectroscopy unable to be used in close to physiological environments. system. NMR, mass spectrometry, fluorescence spectroscopy, and Raman spectroscopy are currently capable of detecting conformational changes in proteins in solution. However, the NMR method is only suitable for samples with a molecular weight of less than 40kDa, and its sensitivity is low. The analysis concentration usually needs to reach the mM level, and the data processing is complicated and the analysis cost is high. Mass spectrometry can study protein conformation according to the charge valence distribution, and has high sensitivity, real-time monitoring and quantitative analysis, but commonly used ESI-MS and improved cold spray ionization CSI-MS are all for the analysis and detection of gaseous ions , the information given does not quite accurately characterize the true conformation of the protein in solution. Fluorescence resonance energy transfer (FRET) is the most widely used fluorescence method, which requires the introduction of larger volumes of donors and acceptors, which will have a certain impact on protein structure and function, and the fluorescence method is also widely used now (such as The effect of FRET is relatively sensitive in the range of 10nm, and it is difficult to apply beyond this limit). On the contrary, Raman spectroscopy is simple and easy, free from water interference, without labeling and special preparation of the sample to be tested, and can provide the intrinsic information of the sample such as the molecular composition and structure of the substance, and obtain rich fingerprint peaks. It has been applied in cells, tissues and even living bodies. However, the traditional detection method based on Raman spectroscopy has low sensitivity, and the spectral peaks of natural biomolecules overlap and interfere seriously, which seriously limits the further application of Raman method.

Contents of the invention

The purpose of the present invention is to provide a method for specifically labeling and detecting proteins by using a bio-orthogonal Raman reporter group to solve the problems in the prior art.

In order to improve the sensitivity of Raman spectroscopy, researchers have developed various enhancement techniques in recent years, such as surface-enhanced Raman (SERS). SERS is an enhancement phenomenon of Raman signals on metal surfaces such as Au and Ag. Generally, an enhancement effect of 10 ⁶ -10 ¹² can be achieved, and a sensitivity similar to that of fluorescence spectra can be achieved. On the other hand, in order to solve the problem of overlapping Raman spectrum peaks of biomolecules, the inventors of the applicant have studied the strategy of orthogonal Raman labeling, that is, introducing Raman signal orthogonal groups (such as alkynyl groups) on biomolecules. , azide, CD, etc.), their Raman signals are in the silent region of cell Raman signals (1800-2800cm ^-1 ).

In order to achieve the above object, the method of the present invention using a bio-orthogonal Raman reporter group to specifically label and detect proteins comprises the following steps:

a) preparing unnatural amino acids carrying bioorthogonal Raman reporter groups;

b) inserting the non-natural amino acid prepared in step a) into the target protein by using gene extension technology, so as to realize bio-orthogonal Raman reporter labeling of the target protein;

c) using bio-orthogonal Raman technology to detect the protein obtained in step b).

Preferably, the above-mentioned bioorthogonal Raman reporter group is an alkynyl group, azide group, cyano group or a carbon-deuterium bond.

Preferably, the above method further includes utilizing the surface enhancement effect of gold nano-ions.

Preferably, the gold nanoparticles AuNPs utilizing the surface enhancement effect of gold nanoparticles are prepared by using sodium citrate reduction method.

Preferably, the final concentration of HAuCl ₄ used in the sodium citrate reduction method is 0.25 mM, and the final concentration of sodium citrate is 0.01% (w/w).

Preferably, the above-mentioned method for specifically labeling and detecting proteins using a bioorthogonal Raman reporter group may further include increasing the affinity of proteins to AuNPs.

Further preferably, increasing the affinity between the protein and AuNPs includes using a cysteine-containing tag, a methionine-containing tag or a small molecule containing a sulfhydryl group on the chemical modification of the protein.

Another object of the present invention is to provide a method for detecting protein conformation changes in vitro using bio-orthogonal Raman technology, comprising the following steps:

a) Under different solution conditions, using the above-mentioned method of using a bioorthogonal Raman reporter group to specifically label and detect proteins to detect protein conformation changes;

b) On the basis of detecting the protein conformation change based on the step a), the relationship between the protein conformation change and the environment change is studied.

Another object of the present invention is to provide a method for detecting protein conformation changes in situ in living cells using bio-orthogonal Raman technology, comprising the following steps:

a) detecting protein conformation changes on the surface of living cells by using the above-mentioned method of using a bioorthogonal Raman reporter group to specifically label and detect proteins;

b) On the basis of detecting protein conformational changes based on step a), at the level of living cells, in situ research on the relationship between protein conformational changes and its protein activity regulated by stimulation.

Preferably, the aforementioned stimuli include growth factor stimulation, ligand stimulation or environmental change stimulation.

In the present invention, the bio-orthogonal Raman reporter group is used to specifically label and detect proteins, using gene extension technology and carrying a bio-orthogonal Raman reporter group (including but not limited to alkynyl, azide, cyano, carbon Deuterium bond, etc.) non-natural amino acid technology to realize the method of bioorthogonal Raman reporter group labeling of target protein.

A method for detecting bioorthogonal Raman reporter groups on specific proteins in solution is realized by utilizing the surface enhancement effect of gold nano-ions. The method is efficient, sensitive, and specific for the target protein.

Using bioorthogonal Raman labeling combined with the surface enhancement effect of gold nano-ions, a method for detecting specific proteins at the level of living cells is realized. The method has good biocompatibility, simple operation, high efficiency, sensitivity and target protein specificity.

The present invention also provides a method for in vitro detection of protein conformation changes using a bio-orthogonal Raman strategy; using bio-orthogonal Raman reporter groups such as alkynyl groups, combined with the surface enhancement effect of gold nano-ions, to detect protein conformation under different solution conditions The method of change; based on the detection of protein conformation change based on bioorthogonal Raman, a new method for studying the relationship between protein conformation change and environmental change.

Using cysteine-containing tags to increase the affinity of proteins with AuNPs, of course, including other methods of increasing affinity for SERS-enhanced bioorthogonal Raman strategy to detect protein local conformational changes, including but not limited to methionine-containing tags or Small molecules containing sulfhydryl groups on proteins chemically modified.

The present invention also provides a method for detecting protein conformation changes in situ in living cells using a bio-orthogonal Raman strategy; using a bio-orthogonal Raman reporter group such as alkynyl, combined with the surface enhancement effect of gold nano-ions, to detect proteins on the surface of living cells Method of conformational change; on the basis of detecting protein conformational changes based on bioorthogonal Raman, at the level of living cells, in situ research on protein conformational changes and the regulation of protein activity by stimuli (growth factors, ligands, and environmental changes, etc.) new method.

The invention provides a method for specifically labeling and detecting proteins using bio-orthogonal Raman reporter groups, a method for detecting protein conformation changes in vitro using bio-orthogonal Raman technology, and using bio-orthogonal Raman technology in situ in living cells A method for detecting protein conformational changes, through gene codon amplification technology and SERS enhanced methods, to achieve rapid, sensitive and efficient detection of conformational changes of proteins with bio-orthogonal groups under different microenvironment or activity conditions , especially to realize the in situ detection of protein conformation changes in living cells, and then analyze the link between protein conformation and function. This technology is based on the method of introducing a bioorthogonal Raman reporter group at a specific site of the protein and the theoretical basis that the molecular vibration signal of the Raman group is very sensitive to changes in the microenvironment and will shift with changes in the environment. Detection of protein conformational changes in solution and in situ and sensitive detection of protein conformational changes in living cells.

The above-mentioned method of the present invention is suitable for bio-orthogonal Raman detection of surface protein conformation changes in protein solution and living cell physiological conditions. The above method of the present invention has the characteristics of being simple and easy, the system is close to physiological conditions, high sensitivity, small interference of orthogonal Raman signals, simple data processing, no restriction on protein molecular weight, and suitable for various protein systems.

Description of drawings

Fig. 1 is the detection of protein conformation change based on bioorthogonal Raman reporter group (alkyne group);

Figure 2 is the Raman shift of the 35th alkynyl group of HdeA protein at pH 7;

Figure 3 is the Raman shift of the 35th alkynyl group of HdeA protein under the condition of pH 2;

Figure 4 is the Raman shift of the 58-position alkynyl of HdeA protein under the condition of pH 7;

Figure 5 is the Raman shift of the 58-position alkynyl of HdeA protein under the condition of pH 2;

Figure 6(a) is the Raman spectrum of EGFR (wt-EGFR) that has not been mutated on the cell;

Figure 6(b) is the Raman spectrum of EGFR with mutation at position 356 on the cell and inserted into Penk but not stimulated by EGF;

Fig. 6(c) is the Raman spectrum of EGFR stimulated by EGF with the mutation at position 356 on the cell and Penk inserted.

detailed description

In order to make the object, technical solution and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The invention uses gene amplification technology to insert non-natural amino acids containing orthogonal groups at specific sites on the protein, and uses gold nano-ions to perform plasma enhancement on Raman signals, so as to realize efficient, sensitive and rapid detection of proteins. Because the Raman signal of the group will be affected by the micro-environment near the group (such as the type of solution, the type of the group, the electrical properties, etc.), and the Raman peak position will shift, and the Raman orthogonal reporter group will be realized. detection of changes in its microenvironment. The change of the microenvironment of the Raman orthogonal reporter group is associated with the conformational change of the protein, which reflects the transformation of the protein conformation. When the protein is close to the gold nanoparticle (less than 10nm), the Raman signal of the bioorthogonal Raman reporter group (alkyne group here) is detected under different conditions to obtain information on the conformational change of the target protein. See Diagram 1 for details.

The strategy of bioorthogonal Raman labeling is to introduce Raman signal orthogonal groups (such as alkynyl, azide, CD, etc.) -2800cm ^-1 ). This strategy solves the problem of overlapping Raman spectral peaks of biomolecules and greatly simplifies the analysis process of Raman data. The gene codon amplification technology utilizes the property that the stop codon TAG in eukaryotes does not encode amino acids and has a low probability of use, and introduces a screened aminoacyltransferase-tRNA pair orthogonal to TAG and unnatural amino acids, so that cells will TAG mutation site of unnatural amino acid insertion protein. Using gene amplification technology to insert non-natural amino acids containing Raman orthogonal groups into proteins for bioorthogonal Raman detection.

Example 1 Detection of conformational changes of alkyne-containing HdeA protein in solution at different pH

HdeA is a molecular chaperone protein in the bacterial membrane stroma. In the physiological environment of pH 7, HdeA exists in the form of homodimerization; while in the physiological environment of pH 2, HdeA depolymerizes to expose its active site and bind to its substrate protein. In the physiological environment of pH 2, HdeA is in a disordered state, and its structure and conformational changes cannot be studied by X-ray crystallography. Penk is a lysine derivative containing an alkyne group (as shown in the figure below), and the orthogonal Penk-MbPylRS/tRNA can be used to insert Penk into the TAG site of the protein.

Synthesis of AuNPs: Gold nanoparticle (AuNPs) sol was prepared by sodium citrate reduction method, wherein the final concentration of HAuCl ₄ was 0.25mM, and the final concentration of sodium citrate was 0.01% (w/w). In boiling ultrapure water, add HAuCl ₄ and sodium citrate under vigorous stirring, and stop heating after 30 min.

Expression and purification of alkyne-containing HdeA: A tag containing multiple cysteines (GCCPGCCGGSGS) was introduced after the N-terminal signal peptide of the bacterial HdeA protein, and a His6 tag was introduced at the C-terminal. The corresponding codon sequence of the 35th or 58th amino acid is mutated into TAG, and the non-natural amino acid Penk containing an alkyne group is introduced. Insert the modified HdeA gene sequence into the pBAD vector. Protein expression was performed using E. coli, and expression was induced using arabinose in the presence of unnatural amino acids. The expressed protein was then purified using a Ni column. An HdeA protein containing an alkyne group is obtained.

Incubation of AuNPs and HdeA: 0.1 mg/ml of alkyne-containing HdeA was added to the AuNPs solution, and the cysteine label was used to bind to the AuNPs. After incubating at room temperature for 0.5 h, 10x buffer solution was added to make the final buffer solution concentration 1xPBS (pH 7) or sodium citrate buffer solution (10mM, containing 150mM NaCl, pH 2). The Raman spectrum of the solution was then detected using a Horiba Raman microscope.

Raman test results are as follows:

1. Protein HdeA (alkynyl at 35th position), pH 2 to 7

As shown in Figure 2, under the condition of pH 7, HdeA is in the inactive state of homodimerization, and the Raman peak of the alkynyl group at position 35 is a single peak at 2120.4 cm ^-1 . As shown in Figure 3, under the condition of pH 2, HdeA depolymerizes into monomers and gradually forms an activated conformation. At this time, the Raman peak of the 35-position alkynyl group appears a trailing shoulder peak, which can be divided into peaks at 2126.1 cm ^-1 and 2146.7cm ^-1 . Both are shifted toward high wavenumbers relative to 2120.4 cm ^-1 , corresponding to the "beginning to disaggregate" and "nearly activated" conformations, respectively.

2. Protein HdeA (alkynyl at position 58), pH 2 to 7

As shown in Figures 4 and 5, the conformational change of the alkynyl at the 58th position of HdeA is similar to that at the 35th position of HdeA. However, the proportions of "beginning to depolymerize" and "nearly activated" conformations are different. Because the changes of different amino acid positions in HdeA are not completely synchronized in the process of depolymerization and gradually forming an activated conformation, the proportions of "beginning to depolymerize" and "nearly activated" conformations reflected by different amino acid positions are different. The invention provides a method for specifically labeling and detecting proteins using a bio-orthogonal Raman reporter group, capable of studying the presence, speed and proportion of conformational changes at different amino acid sites in the process of protein conformational changes.

Example 2 Detection of conformational changes of cell surface alkyne-containing EGFR protein before and after EGF stimulation

EGFR is an epidermal growth factor receptor protein located on the surface of cell membranes. The EGFR signaling pathway plays an important role in physiological processes such as cell growth, proliferation and differentiation, and is closely related to a variety of cancers. EGFR will dimerize after combining with epidermal growth factor EGF, and EGF mainly exists in the form of monomer after being released.

Synthesis of AuNPs: Prepared by sodium citrate reduction method.

Expression of alkyne-containing EGFR in cells: the codons at positions 210, 250, 356 or 440 of the EGFR protein were mutated to TAG. In the presence of the unnatural amino acid Penk, a plasmid containing the engineered EGFR and Penk- _MbPylRS /tRNA ^PylCUA gene sequence was transiently transformed into HEK293T cells. After 40 hours, the cells were starved for 8 hours with serum-free medium, and then the cells in the experimental group were stimulated with 100 ng/ml EGF solution, and after 15 minutes, the cells were fixed with 4% paraformaldehyde solution. Add 1/10 volume of 10×PBS to the gold sol, quickly add to the cell system and incubate for 1 hour. The Raman spectra of the cells were detected using a Nanophton Raman microscope.

The Raman results are as follows:

As shown in Figure 6(a)-6(c), EGFR on cells not stimulated with EGF (EGF-) mainly exists in the form of monomers, and EGFR on cells stimulated with EGF (EGF+) mainly exists in the form of dimers. The chemical environment (such as electric field, hydrophilicity and hydrophobicity) around the alkyne group at the same position in the two conformations is different, and the position of its Raman signal will shift. Taking position 356 as an example, the unmutated wildtype-EGFR control group had no Raman signal of the alkynyl group, and the Raman signal of the alkynyl group could be detected in the experimental group mutated at position 356 and inserted into Penk. Among them, the EGF-group cells The Raman signal of the alkyne group was at 2122cm ^-1 , and that of the cells in the EGF+ group was at 2126cm ^-1 , that is, the Raman signal of the alkyne group was shifted due to the conformational change of EGFR before and after EGF stimulation. The statistical results of mutations at 4 different sites are shown in the table below, among which sites 250, 356, and 440 have high wavenumber shifts in Raman signals after EGF stimulation, while site 210 has low wavenumber shifts, reflecting the chemistry of different sites in EGFR. Environments undergo different trends in conformational transitions.

Table 1 shows the Raman shifts of alkynyl groups at different sites of EGFR on cells before and after EGF stimulation, as follows:

Table 1

The results of solution and cells showed that the change of Raman signal of the alkyne group inserted into the protein under different conditions can reflect the conformational change of the protein. Bioorthogonal Raman technology enables in situ detection of protein conformational changes.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art may make some changes and improvements without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims (10)

1. A method for using bio-orthogonal Raman reporter group specific labeling and detection of proteins, characterized in that, comprising the steps: a) preparing unnatural amino acids carrying bioorthogonal Raman reporter groups; b) inserting the non-natural amino acid prepared in step a) into the target protein by using gene extension technology, so as to realize bio-orthogonal Raman reporter labeling of the target protein; c) using bio-orthogonal Raman technology to detect the protein obtained in step b). 2. The method according to claim 1, wherein the bioorthogonal Raman reporter group is an alkynyl group, an azide group, a cyano group or a carbon-deuterium bond. 3. The method according to claim 1, further comprising utilizing the surface enhancement effect of gold nano-ions. 4. The method according to claim 3, wherein the gold nanoparticles AuNPs in the surface enhancement effect utilizing gold nano ions are prepared by using a sodium citrate reduction method. 5. The method according to claim ₄ , wherein the final concentration of HAuCl used in the sodium citrate reduction method is 0.25mM, and the final concentration of sodium citrate is 0.01% (w/w). 6. The method of claim 3, comprising increasing the affinity of the protein for AuNPs. 7. The method according to claim 6, wherein said increasing the affinity of the protein to AuNPs comprises using a cysteine-containing label, a methionine-containing label or a small molecule containing a sulfhydryl group on the chemical modification of the protein . 8. A method for detecting protein conformation changes in vitro using bio-orthogonal Raman technology, characterized in that it comprises the steps of: a) under different solution conditions, utilizing the method described in any one of claims 1-7 to detect protein conformational changes; b) On the basis of detecting the protein conformation change based on the step a), the relationship between the protein conformation change and the environment change is studied. 9. A method of using bio-orthogonal Raman technology to detect protein conformation changes in situ in living cells, characterized in that it comprises the following steps: A) utilize the method described in any one in claim 1-7 to detect the protein conformation change of living cell surface; b) On the basis of detecting protein conformational changes based on step a), at the level of living cells, in situ research on the relationship between protein conformational changes and its protein activity regulated by stimulation. 10. The method of claim 9, wherein the stimulus comprises a growth factor stimulus, a ligand stimulus, or an environmental change stimulus.