Method for resolving protein structures using a cryo-electron microscope

Abstract

The present application provides a method for protein structure analysis using cryo-EM, comprising: including a tag in a target protein; combining the target protein including the tag with a scaffold protein to form a complex of the target protein and the scaffold protein; and using a cryo-EM for single particle imaging to analyze the structure of the target protein complexed with the scaffold protein. The scaffold protein is any one of streptavidin, avidin or a derivative thereof. The tag is used to selectively bind to the scaffold protein. The tag is selected from one of the following group: a biotin tag including biotin; a biotinylated protein or polypeptide tag including a protein sequence and biotin covalently linked to the protein sequence; a streptomyces tag (Strep-tag); and a biotinylated or streptococcal labeled antibody, antibody Fab fragment or single chain antibody.

Description

BACKGROUND TECHNICAL FIELD

[0001] The present application relates to the technical field of structural biology, and in particular to a method for resolving protein structure using cryo-electron microscopy.

[0002] Description of Related Art

[0003] The statements herein are provided only to complement the present application and do not necessarily constitute the prior art.

[0004] High-resolution three-dimensional structures of proteins provide important information for understanding the function of proteins (Garcia-Nafria et al., 2020). Classical protein structures are determined using X-ray crystallography and NMR spectroscopy. Depending on the class of protein, different hurdles have to be overcome to produce high-resolution protein structures. In the case of X-ray crystallography, suitable diffracting crystals have to be generated, which requires a large amount of purified protein. In the case of NMR spectroscopy, protein structures can be studied in solution, but have to be labelled with appropriate isotopes in the main chain and side chains. Both structure determination techniques are very time-consuming and can be very expensive. This is particularly true when determining three-dimensional structures of membrane proteins (Li et al., 2021). About one third of the human proteome are membrane proteins. Many of them play a central role in the normal functioning or malfunctioning of cells. Their malfunctioning leads to many serious diseases, and therefore membrane proteins are the targets of most modern drugs. Their importance for modern drug development will further increase (Sriram and Insel, 2018).

[0005] The determination of three-dimensional structures of membrane proteins does come with additional hurdles compared to water-soluble proteins. Membrane proteins first have to be isolated from their natural cell membranes, which is usually done by solubilizing the entire membrane in a detergent, so as to produce detergent-solubilized, molecularly state membrane proteins. Under these conditions, membrane proteins can be purified in a homogeneous form. For X-ray crystallography, detergent-solubilized membrane proteins have to be made to form crystals. In most cases, membrane proteins only produce highly diffracting crystals after severe modifications to their native sequence. Resolving the structure of membrane proteins by NMR spectroscopy faces an additional limitation by the size of the protein: only structures of proteins with a molecular weight below 80 kDa can routinely be solved by currently available techniques (Purslow et al., 2020).

[0006] Recent advances in single-particle imaging using cryo-electron microscopy (cryoEM) have revolutionized membrane protein structural biology (Kühlbrandt, 2014; Garcia-Nafria et al., 2020). Among them, many individual membrane proteins or membrane protein complexes are imaged in detergent-solubilized form or reconstituted into lipid bilayer nanodiscs. From the images of millions of individual particles, 3D structures can be estimated at atomic resolution for the corresponding wild-type membrane protein, i.e. without the need for crystals.

[0007] For some large or symmetric complexes, cryoEM structures at resolutions exceeding 10 Å have been achieved (Greber et al., 2021). However, many drug targets are neither large nor symmetric. Currently, the widespread application of single-particle cryoEM for improving the resolution of membrane protein structures is limited by the following factors:

[0008] (i) Despite significant technical progress, it is still very challenging to perform high-resolution structure determination of asymmetric complexes with molecular weights below 100-kDa at resolutions of 10 Å or better.

[0009] (ii) Another serious problem is protein adsorption at the air-water interface of the sample preparation for electron microscopy. This can lead to preferential protein orientations and protein denaturation, thereby significantly reducing the sample quality for single-particle analysis. SUMMARY

[0010] In view of the above technical problems, one of the purposes of the present application is to provide a method for protein structure determination using cryo-electron microscopy, which is a general method and can solve the problem of structure determination of target proteins with a molecular weight of at least 20 kDa.

[0011] To achieve the above-mentioned purpose, according to an embodiment of the present application, a method for protein structure determination using cryo-electron microscopy is provided. The method comprises:

[0012] including a tag in a target protein;

[0013] binding the target protein including the tag to a scaffold protein to form a complex of the target protein and the scaffold protein; and

[0014] performing single-particle imaging using cryo-electron microscopy to determine the structure of the target protein complexed with the scaffold protein.

[0015] The scaffold protein is any one of streptavidin, avidin or a derivative thereof. The tag is selected to selectively bind to the scaffold protein. The tag is selected from the group consisting of: a biotin tag comprising biotin; a biotinylated protein or polypeptide tag comprising a protein sequence and biotin covalently linked to the protein sequence; a Strep-tag; and a biotinylated or streptococcal labelled antibody, antibody Fab fragment or single chain antibody.

[0016] In embodiments of the application, the Strep-tag is a polypeptide sequence adapted to selectively bind to streptavidin or streptavidin or other derivatives thereof.

[0017] In embodiments of the application, where the tag is the biotin tag, the step of causing the target protein to comprise a tag comprises:

[0018] incorporating the biotin tag in the side chain of a non-canonical amino acid and site-specifically introducing the non-canonical amino acid into the amino acid sequence of the target protein;

[0019] or chemically linking the biotin tag to a specific side chain of an amino acid (such as the epsilon-amino group of lysine or the -SH group of cysteine) and site-specifically introducing the amino acid into the target protein;

[0020] or chemically linking the biotin tag to a specific glycosylation site of the N-terminal portion of the target protein.

[0021] In embodiments of the application, the biotin tag is selectively attached to the target protein by chemical modification or genetic engineering.

[0022] In embodiments of the application, the biotinylated protein or polypeptide tag is selected from the group consisting of:

[0023] a self-labelling protein tag that allows site-specific covalent attachment of a biotin residue to the respective tag protein;

[0024] an acyl carrier protein tag (ACP-tag) or a peptidyl carrier protein tag (PCP-tag); and

[0025] an Avi-tag adapted to be fused to the N- or C-terminus of the target protein or to an exposed loop region and covalently linked to biotin by the Escherichia coli biotinylation enzyme BirA.

[0026] In embodiments of the application, the ACP-tag or PCP-tag and biotin are enzymatically covalently linked by Sfp- and AcpS-PPTase, respectively, using a biotin derivative of CoA as a substrate.

[0027] In embodiments of the application, the self-labeling protein tag is a Snap-tag, Clip-tag or Halo-tag.

[0028] In embodiments of the application, the target protein preferably has a molecular weight of between 20 kDa and 50 kDa or even less. In embodiments of the application, the target protein has a molecular weight of at least 33 kDa or less. In embodiments of the application, the target protein has a molecular weight of at least 20 kDa or less.

[0029] In embodiments of the application, the target protein is a water-soluble protein or a membrane protein.

[0030] In embodiments of the application, when the target protein is a G-protein coupled receptor, GPCR for short, the biotin tag or the biotinylated protein or polypeptide tag is inserted at one of the N-terminus, C-terminus or one of the extracellular loops (i.e. E1, E2, E3) or one of the intracellular loops (i.e. 11, 12, 13, 14).

[0031] In embodiments of the application, when the target protein is a GPCR, the anticalin or Ig-type or single-chain antibody is selectively linked at one of the N-terminus, C-terminus or one of the extracellular loops (i.e. E1, E2, E3) or one of the intracellular loops (i.e. 11, 12, 13, 14).

[0032] In embodiments of the application, the biotin tag, the biotinylated protein or polypeptide tag or the Strep-tag is fused at the intracellular loop I4 of the GPCR, thereby stabilizing the bound streptavidin or streptactin in a rigid structure.

[0033] In embodiments of the application, the biotin tag, the biotinylated protein or polypeptide tag or the Strep-tag is fused at one of the extracellular loops (i.e. E1, E2, E3) or one of the intracellular loops (i.e. 11, 12, 13, 14) of the GPCR, thereby stabilizing the bound streptavidin or streptactin in a rigid structure.

[0034] In embodiments of the application, when the target protein is a complex of a prototypical GPCR with an intracellular signaling protein, the tag can be introduced to bind the target protein to the streptavidin or derivative thereof as a scaffold protein by:

[0035] 1) inserting the biotin tag, the biotinylated protein or polypeptide tag at one of the N-terminus, C-terminus or one of the extracellular loops or one of the intracellular loops of the prototypical GPCR;

[0036] 2) selectively attaching an anticalin or an Ig-type or single chain antibody at one of the N-terminus, C-terminus or one of the extracellular loops or one of the intracellular loops of the prototypical GPCR;

[0037] 3) fusing the biotin tag or the biotinylated protein or polypeptide tag into the sequence of the intracellular signaling protein;

[0038] 4) selectively binding a biotinylated antibody to the intracellular signaling protein; or

[0039] 5) selectively binding a biotinylated anticalin to the intracellular signaling protein.

[0040] In embodiments of the application, the intracellular signaling protein is selected from any one of:

[0041] a heterotrimeric G protein or a mini G protein adapted to bind to a GPCR activated by an agonist;

[0042] a G protein coupled receptor kinase (GRK) adapted to bind to and phosphorylate an active GPCR;

[0043] an arrestin adapted to bind to a phosphorylated GPCR; and

[0044] a peptide sequence mimicking a region of the G protein or arrestin that binds to the receptor.

[0045] In embodiments of the application, when the target protein is a neuropeptide 1 receptor (NK1R), the tag is a biotinylated Halo-tag and the scaffold protein is streptavidin, a tetrameric NK1R is assembled on the streptavidin via the biotinylated Halo-tag, i.e. a complex SA(HaloTag-NK1R) is formed n adapted to resolve the structure of the NK1R by cryo-EM, wherein n is an integer between 1 and 4.

[0046] In embodiments of the present application, the biotinylated Halo-tag is inserted in the third intracellular loop structure IL3 of the NK1R, and the amino acid sequence from position 227 to 237 is deleted.

[0047] In embodiments of the present application, before the step of incorporating a tag into the target protein, a HaloTag-PEG4-biotin ligand is synthesized, and a stable ester bond is formed between the HaloTag-PEG4-biotin ligand and the HaloTag protein to form a biotinylated Halo-Tag tag.

[0048] In embodiments of the present application, the molecular formula of the HaloTag-PEG4-biotin ligand is C 31 H 57 ClN4O9S, with a molecular weight of 697, which has the following chemical structure:

[0049]

[0050] In embodiments of the present application, the terminal -Cl of the HaloTag-PEG4-biotin ligand can be replaced by Asp106 of the HaloTag protein in an affinity reaction to form a stable ester bond, thereby forming a biotinylated Halo-tag tag.

[0051] In embodiments of the present application, before the step of incorporating a tag into the target protein, the method comprises molecular modeling for finding and adjusting the appropriate insertion position of the biotin tag or biotinylated protein or polypeptide tag or the strep-tag in the target protein sequence to obtain the best structural rigidity between the target protein and the scaffold protein.

[0052] In embodiments of the present application, during the molecular modeling process, additional spacer amino acids are inserted into the amino acid sequence of the target protein, or functionally unrelated flexible amino acid sequences are removed from the amino acid sequence of the target protein.

[0053] In embodiments of the present application, the PEG4 spacer in the HaloTag-PEG4-biotin ligand is further shortened or expanded to obtain the best structural rigidity between the scaffold protein and the target protein.

[0054] The advantages of the method for analyzing protein structure using a low-temperature electron microscope according to embodiments of the present application are summarized as follows:

[0055] In the method of the present application, the target protein is made to comprise a tag. The target protein comprising the tag is bound to a scaffold protein to form a complex. Then single particle imaging is performed using cryo-electron microscopy to determine the structure of the target protein. The tag is capable of selectively binding to the scaffold protein. In this way, the method is versatile for structure determination of a variety of soluble proteins or membrane proteins. Therefore, the target proteins are not limited to their symmetry structure and large molecular weight. In contrast, the method of the present application can achieve structure determination of target proteins with a molecular weight as low as at least 20 kDa, and the determination result is not affected by the symmetry of the structure of the target protein.

[0056] Another object of the present application is to provide a use of a HaloTag-PEG4-biotin ligand in the above-mentioned method for determining the structure of a protein using a low-temperature electron microscope. The HaloTag-PEG4-biotin ligand has a molecular formula of C31H57ClN4O9S and a molecular weight of 697, and has the following chemical structure:

[0057]

[0058] In the embodiments of the present application, Asp106 of the HaloTag protein is adapted to replace the terminal -Cl of the HaloTag-PEG4-biotin ligand in an affinity reaction to form a stable ester bond, thereby forming a biotinylated Halo-Tag tag.

[0059] The advantages of the HaloTag-PEG4-biotin ligand according to the embodiments of the present application are summarized as follows:

[0060] The terminal -Cl of the HaloTag-PEG4-biotin ligand can be replaced by Asp106 of the HaloTag protein in a nucleophilic reaction to form a stable ester bond, thereby forming a biotinylated Halo-Tag. The biotinylated Halo-Tag is capable of connecting the target protein with the scaffold protein to form a protein complex, which is suitable for single particle cryo-EM to determine the structure of the target protein. The length of the PEG4 spacer in the HaloTag-PEG4-biotin ligand can be changed to minimize the flexibility between the HaloTag protein and the streptavidin scaffold protein.

[0061] BRIEF DESCRIPTION OF DRAWINGS

[0062] The present application will be described below with reference to the accompanying drawings, in which:

[0063] Figures 1A-1C shows the formation of a tetramer of a target protein or protein complex on a scaffold protein for single particle cryo-electron microscopy. In particular, Figure 1AA schematic showing a prototypical G protein coupled receptor (GPCR) consisting of seven transmembrane helices (rectangles 1, 2,... 7) and helix 8 (rectangle 8) at the C-terminus inside the cell. The extracellular N-terminus contains a biotinylation tag; as Figure 1B A schematic showing a prototypical GPCR in complex with an intracellular signaling protein; and Figure 1C A schematic showing a scaffold protein with a protein of interest (POI) bound specifically to each of the four scaffold binding sites via its tag.

[0064] Figure 2A A structural model of SA-(NK1R-halotag)4 is shown, including a tetrameric streptavidin SA (center) and four bound biotinylated HaloTag protein molecules. The structural model was obtained by computer modeling.

[0065] Figure 2B Intrinsic mobility of the protein complex calculated from an anisotropic network model (ANM) analysis is shown.

[0066] Figure 3A A structural model of SA(HaloTag)4 is shown, including a tetrameric streptavidin (SA center, orange) and four bound biotinylated HaloTag protein molecules (green), one HaloTag protein per SA subunit.

[0067] Figure 3B Intrinsic mobility of the complex calculated from an ANM analysis is shown.

[0068] Figure 4A Purified HalotagPEG-biotin ligand analyzed by HPLC: the wavelength of light absorption is 210 nm and the retention time is 10 minutes.

[0069] Figure 4B LC-MS of HalotagPEG-biotin ligand. MS: m / z calculated. For C 31 H 58 ClN4O9S + : 697.36 (100%), 698.36 (36.6%), 699.36 (41%), 719.3 (12.13%), 720.3 (4.41%).

[0070] Figure 5A Size exclusion chromatography of streptavidin-halotag protein complex is shown. Figure 5B SDS-PAGE of streptavidin tetramer (streptavidin), halotag protein (halotag), size exclusion chromatography fraction 1, and fraction 2 are shown.Figure 5A ).

[0071] Figure 6 An electron micrograph of a negatively stained preparation of streptavidin-halotag protein complex separated by size exclusion chromatography is shown (scale bar: 50 nm). Figure 5A

[0072] Figure 7A A representative cryo-electron micrograph of streptavidin-halotag protein complex is shown (cryo-electron microscope was operated at 300 kV; scale bar: 50 nm). Figure 7B A representative two-dimensional class average image of streptavidin-halotag protein complex is shown.

[0073] Figures 8A-8B Resolution estimation of cryo-electron micrographs of streptavidin-halotag protein complex is shown. Specifically, Figure 8A A Fourier shell correlation (FSC) plot is shown indicating a corrected resolution of 3.6; Figure 8B is a three-dimensional map of streptavidin-halotag protein complex after non-uniform refinement.

[0074] Figure 9A Size exclusion chromatography of streptavidin-NK1R-halotag complex is shown; and

[0075] Figure 9B SDS-PAGE of streptavidin-NK1R-halotag complex, NK1R-Halotag corresponding to size exclusion chromatography fractions 1 to 4 is shown Figure 9A DETAILED DESCRIPTION

[0076] To further illustrate the present application, experiments detailing the method of using cryo-electron microscopy to resolve protein structure are described below. It is to be understood that the following examples are provided by way of illustration and not by way of limitation.

[0077] DEFINITIONS

[0078] ​​The indefinite articles “a” or “an,” as well as “the” are used herein to include the plural form as well, unless the context clearly indicates otherwise. The term “includes” as used herein is meant to be equivalent to the term “comprising” and therefore does not exclude other elements or steps. Furthermore, the terms first, second, third, etc. as used in the description and claims are used to distinguish between similar elements, not necessarily in a chronological or sequential order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the application described herein are capable of operating in other sequences than the one described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the application. Unless specifically defined herein, all terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this application belongs.

[0079] As used herein, the term “target protein” or “protein of interest (POI)” refers to a protein whose structure is to be determined.

[0080] “Scaffold protein” as used herein refers to a protein having multiple protein binding domains that specifically bind to a tag or modified tag attached to a protein whose structure is to be determined.

[0081] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues, variants, and synthetic analogs thereof.

[0082] Biotin is hexahydro-2-oxo-lH-thieno(3,4-d)imidazole-4-pentanoic acid. It is also known as vitamin H and coenzyme R, is a water-soluble vitamin, also belonging to the vitamin B family, B7. It is an essential substance for the synthesis of vitamin C and is indispensable for the normal metabolism of fats and proteins.

[0083] Streptavidin is a protein complex composed of four identical protein subunits. The protein molecular weight of this tetramer is 52 kDa and can be produced either by purification from Streptomyces avidinii bacteria or by expression of the heterologous protein in E. coli. The streptavidin homotetramer has an extremely high affinity for biotin (also known as vitamin B7 or vitamin H). The dissociation constant (Kd) is about 10 -14 mol / L, the binding of biotin to streptavidin is one of the strongest non-covalent interactions known in nature. Due to the resistance of the streptavidin-biotin complex to organic solvents, denaturing agents (e.g. guanidinium chloride), detergents (e.g. SDS, Triton X-100), proteolytic enzymes, as well as to extreme temperatures and pH values, streptavidin is widely used in molecular biology, bioanalysis, and biotechnology.

[0084] In the present context, water-soluble proteins of interest having a molecular weight below 50 kDa include enzymes, transcription factors and transport proteins, found free in cellular compartments such as cytoplasm, nucleus or endoplasmic reticulum.

[0085] Integral membrane proteins, in the present context simply referred to as "membrane proteins", are proteins that are embedded in the phospholipids that make up the bilayer structure of the cell membrane. Membrane proteins perform specific functions that are essential for the normal functioning of the cell. These specific functions include translocation of molecules and ions into and out of the cell, detection of extracellular signals and transmission of them into the cell. Extracellular sites of membrane proteins are often glycosylated, enabling the cell to be recognized by other cells.

[0086] G-protein coupled receptors (GPCRs) are integral membrane proteins that transmit physical and chemical signals from the extracellular space into the cell. The chemical signals recognized by GPCRs range from small molecules such as neurotransmitters to hormones and proteins. There are approximately 800 representatives of GPCRs in the human proteome that represent the largest family of membrane proteins. There are five different classes of GPCRs in humans. Class A rhodopsins contains the largest number of GPCRs, approximately 700 representatives. About half of these are olfactory receptors. Typical class A GPCRs have a molecular weight in the range of 30 to 40 kDa. Each receptor contains a transmembrane domain consisting of seven alpha-helices. The extracellular N-terminus is often glycosylated. Specific conserved glycosylation sites can be selectively biotinylated by a one-step chemical reaction. The intracellular C-terminus often contains an alpha-helix that is connected to the intracellular side of the plasma membrane by a lipid anchor. GPCRs are among the most important targets for small molecule drugs.

[0087] The neurokinin 1 receptor (NK1R) is the major receptor for tachykinins such as substance P. NK1R belongs to class A GPCRs. It couples to Gq and Gs protein signaling pathways.

[0088] The terms "fusion" and "fused" are used interchangeably herein as "bound", "conjugated", "linked", and in particular refer to "genetic fusion", e.g. by recombinant DNA technology, and to "chemical and / or enzymatic binding" resulting in stable covalent bonds.

[0089] The term "protein complex" or "complex" or "assembled protein" as used herein refers to a group of two or more bound macromolecules, at least one of which is a protein.

[0090] The term "bound" refers to any interaction, whether direct or indirect. A direct interaction indicates that there is contact between the binding partners. An indirect interaction refers to any interaction that causes the interaction partners to interact in a complex of two or more molecules.

[0091] As used herein, the term "antibody" refers to an immunoglobulin (IgG) molecule or a molecule containing an immunoglobulin (Ig) domain that specifically binds to an antigen. The antibody can be a whole immunoglobulin from a natural source or from a recombinant source and can be an immunoreactive portion of a whole immunoglobulin. IgG antibodies are typically composed of four polypeptide chains.

[0092] As used herein, the term "anticalin" refers to a protein engineered with new antibody-like binding function of a lipocalin.

[0093] As used herein, the term "label" or "tag" refers to a detectable label described herein that has been isolated or purified that allows detection, visualization and / or isolation, purification and / or immobilization of a (poly)peptide or protein. It is intended to include any label / tag known in the art for these purposes.

[0094] The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. The wild type gene is the most commonly observed gene in a population and thus the "normal" or "wild type" form of a gene can be arbitrarily designed. In contrast, the term "modified", "mutated", "derivative" or "variant" refers to a sequence, post-translational modification and / or functional characteristic (i.e. altered characteristic) that is displayed when compared to the wild type. Type gene or gene product or modified gene or gene product.

[0095] As used herein, the term "vector", "vector construct", "expression vector" or "gene transfer vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule with which it is associated, and includes any of the vectors known to those skilled in the art, including any suitable type, including but not limited to a plasmid vector, a cosmid vector, a phage vector (e.g. lambda phage), a viral vector (e.g. adenoviral, AAV or baculoviral vector) or an artificial chromosome vector (such as a bacterial artificial chromosome) (BAC), a yeast artificial chromosome (YAC) or a P1 artificial chromosome (PAC).

[0096] Here, we present a new technology to solve the structure of a target protein with a molecular weight as low as at least 20 kDa. It is generic, i.e. it can be used in the same way for many different target proteins. The target protein for structure determination contains a tag that binds selectively to streptavidin, avidin or a modified form thereof (Figure 1). The tag of interest is typically a protein or short peptide sequence that can be covalently attached to biotin. Alternatively, a so-called strep-tag can be used, which is a short peptide sequence that can bind selectively to streptavidin or as a modified form of streptavidin, streptavidin-XT. Different tags of interest are summarized in Table 1.

[0097] Table 1

[0098]

[0099]

[0100]

[0101] In general, tags can be divided into two groups summarized in Table 1.

[0102] (1) biotin tags directly attached to the target protein, or biotinylated protein or polypeptide tags fused to the target protein sequence.

[0103] Fusion proteins of interest comprise one of the following tags:

[0104] (i) biotin contained in the side chain of a non-canonical amino acid, wherein the non-canonical amino acid is site-specifically introduced into the amino acid sequence of the target protein (Kim et al., 2013).

[0105] (ii) biotin chemically linked to a specific glycosylation site at the N-terminus of the target protein (Bieri et al., 1999). More specifically, glycosylation consensus sites near the extracellular N-terminus are conserved among sequenced GPCRs, and glycosylation has been demonstrated for several receptors. This feature can be exploited for biotinylation using carbohydrate-specific chemistry, thereby restricting the biotin tag to the extracellular domain of the receptor.

[0106] (iii) self-labeling protein tags such as Snap-tag, Clip-tag or Halo-tag, allowing site-specific covalent attachment of biotin residues to the respective protein tag (Los et al., 2008; Reymond et al., 2011).

[0107] (iv) acyl carrier protein (ACP) or peptidyl carrier protein (PCP) tags, which can be site-specifically labeled with biotin using Sfp and AcpS phosphopantetheinyl transferases (PPTases), respectively (George et al., 2004; Vivero-Pol et al., 2005). The ACP and PCP tags can be replaced by the 12-residue peptide tags Al and S6, which can be site-specifically labeled with biotin on the fusion protein by Sfp- and AcpS-PPTases, respectively (Zhou et al., 2007).

[0108] (v) A streptag (SEQ ID NO: 1 : WSHPQFEK) consisting of an 8 amino acid peptide (https: / / en.wikipedia.org / wiki / Strep-tag; Schmidt & Skerra, 2007) can be used, which binds strongly to one of the four identical binding sites of Strep-Tactin, a modified version of streptavidin. In total four streptag fused to the respective target protein will bind to one Strep-Tactin molecule. Alternatively, a twin streptag (SEQ ID NO: 2:

[0109] WSHPQFEKGGGSGGGSGGSHPQFEK) can be used, which binds with higher affinity to Strep-Tactin and with sub-nanomolar affinity to Strep-Tactin-XT, another modified version of streptavidin. The streptag can also bind to streptavidin, but with lower affinity compared to Strep-Tactin. Here, one twin streptag sequence binds to two subunits of Strep-Tactin-XT, Strep-Tactin or streptavidin (https: / / www.iba-lifesciences.com; Palmer I et al., 2007), i.e. in this case only two twin-strep-tag sequences can bind to one streptavidin, Strep-Tactin or

[0110] Strep-Tactin-XT molecule.

[0111] (vi) Another option is the 15 amino acid Avitag, which can be fused to the N or C terminus of the target protein or to an exposed loop region and which can be covalently linked to biotin using the E. coli biotinylation enzyme BirA (Fairhead & Howarth, 2015).

[0112] The above mentioned tags provide the additional advantage that they can be used for affinity purification of the target protein (https: / / en.wikipedia.org / wiki / Strep-tag; Lin et al., 2020).

[0113] (2) Biotinylated or streptag-labeled antibodies, antibody Fab fragments or single chain antibodies (nanobodies) or anticalins (Gebauer & Skerra, 2020) that selectively bind to the target protein.

[0114] In this approach, a biotinylation or streptavidin tag sequence is covalently attached to an Ig-type antibody, antibody fragment or nanobody that binds with high affinity and selectivity to a native or genetically introduced sequence or structural motif in the POI. This approach is of particular interest if a native, unmodified POI shall be assembled to the scaffold protein streptavidin or its derivatives.

[0115] Figures 1A-1C The possibility to position biotinylation and streptavidin tags in the context of membrane proteins such as water-soluble proteins or GPCRs is described. GPCRs are the largest class of human membrane proteins and one of the most important drug targets (Zhou et al., 2019; Sriram and Insel, 2018). As most GPCRs comprise S-palmitoylation at some cysteine residue in the C-terminal region (Qanbar and Bouvier, 2003; Adachi et al., 2019; Patwardhan et al., 2021), the intracellular loop I4 between transmembrane helix 7 and the S-palmitoylation site is of particular interest for placing a biotin / Strep-tag (or biotinylated Al and S6 tags) as it can stabilize the bound streptavidin / streptactin in a rigid structure. Similarly, a biotin / Strep-tag placed in one of the extracellular loops El, E2, E3 or one of the intracellular loops Il, I2, I3, I4 can keep the bound streptavidin / streptactin in a rigid structure that will facilitate the structural elucidation of the target GPCR.

[0116] Figure 1A A schematic representation of a prototype G protein-coupled receptor (GPCR) consisting of seven transmembrane helices (rectangles 1, 2,... 7) and helix 8 (rectangle 8) from the intracellular C-terminus is shown. The C-terminal part of the GPCR can be connected to the intracellular side of the lipid bilayer nanodisc via a lipid anchor. In this illustration, the extracellular N-terminus comprises a single tag (e.g., a biotinylation tag) that can selectively bind to one of the four different binding sites of the scaffold protein directly or after post-translational modification (as shown in Figure 1C Typically, the target GPCR comprises a single protein or peptide tag that can be inserted into the N-terminus, the C-terminus or an extracellular (El, E2, E3) or intracellular (Il, I2, I3, I4) loop structure. Alternatively, an Ig-type or single-chain antibody that selectively targets the extracellular or intracellular side of the GPCR can act as a biotinylation tag to bind streptavidin or streptavidin-related scaffolds. Potential protein and peptide tags are listed in Table 1.

[0117] Figure 1BA schematic showing a prototypical GPCR complexed with intracellular signaling proteins, such as: (i) a heterotrimeric G protein or a mini G protein adapted to bind to an agonist-activated GPCR; (ii) a G protein-coupled receptor kinase (GRK) adapted to bind to and phosphorylate an active GPCR; (iii) an arrestin protein adapted to bind to a phosphorylated GPCR; (iv) a peptide mimicking a G protein or arrestin protein sequence in the region that binds to the receptor.

[0118] Different approaches can be taken to introduce a tag into such GPCR / signaling protein complexes to enable these complexes to bind to a streptavidin or streptavidin-like scaffold protein: (i) using a GPCR comprising a tag as described in ( Figure 1A ); (ii) attaching a specific tag to the signaling protein or signaling peptide sequence, in which, in some embodiments, the specific tag is a biotinylated protein or polypeptide tag fused to the target protein sequence; (iii) using a biotinylated anticalin or antibody that targets the signaling protein bound to the GPCR. Figure 1C A schematic showing a scaffold protein comprising a protein of interest (POI) that is specifically bound to each of the four scaffold binding sites via a tag (e.g., a biotinylated tag). The scaffold protein can be streptavidin or avidin that specifically binds to the biotin of the tag. Alternatively, an 11-amino acid streptococcal tag can be used that selectively binds to a scaffold protein streptactin (a modified form of streptavidin). The POI corresponds to a GPCR with an additional tag as described in ( Figure 1A ), or to a GPCR / signaling protein complex with an additional tag as described in ( Figure 1B ).

[0119] The new approach using GPCRs assembled on a streptavidin template via halotags to elucidate the structure of the corresponding GPCR by single-particle cryo-EM was demonstrated using the neurokinin 1 receptor (NK1R) as a prototypical GPCR. The high-resolution structure of the human NK1 receptor (NK1R) in complex with a small molecule antagonist therapeutic has recently been solved by X-ray crystallography and NMR spectroscopy ( , 2019; Chen et al., 2019). Here, NK1R serves as a test case to compare the cryo-EM structure of different streptavidin-(NK1R-halotag)4 structures to the published NMR and X-ray structures.

[0120] Molecular modeling is a very important step in finding the right place for the biotinylation tag or the streptavidin tag in the protein sequence of the GPCR. In a first step, homology computer modeling will be used to build a 3D structure model of any GPCR, the structure of which has to be solved experimentally by the new cryo-electron microscopy method. In a second step, further computer modeling will be used to insert the biotinylation tag of interest into one of the positions of the GPCR as schematically shown in Figures 1A-1C The exact insertion in the protein sequence will be adjusted by computer modeling to obtain the best structural rigidity between the streptavidin or streptavidin template and the GPCR, if necessary, by inserting additional spacer amino acids or removing functionally irrelevant flexible amino acid sequences from the original GPCR amino acid sequence as well as from the amino acid sequence of the tag. Molecular dynamics simulations will be used to probe the flexibility / rigidity of the target protein with respect to the streptavidin template. These simulations will help to optimize the position of the biotin on the target protein to achieve the best accessibility for binding to streptavidin with the lowest mobility. Figures 2A-2B An example of the (HaloTag-biotin)4-streptavidin system is given in

[0121] Figure 2A A structural model of SA-(NK1R-halotag)4 is shown, including a tetrameric streptavidin SA (center) and four bound biotinylated HaloTag protein molecules, each biotinylated HaloTag protein molecule inserted into the third intracellular loop I3 structure (see Figure 1A ), removing the amino acid sequence between amino acids 227 and 237 from the NK1 receptor.

[0122] Figure 2B The intrinsic mobility of the protein complex calculated from anisotropic network model (ANM) analysis is shown. The green vector length correlates with the relative amplitude of motion of the different protein domains. There is one overall motion of each NK1 receptor subunit relative to the single HaloTag protein relative to the SA template. Only one NK1 receptor is incorporated into the POPC lipid bilayer, not shown for clarity.

[0123] It is clear that the method described in the foregoing can easily be adapted to solve the structure of a water-soluble protein as target.

[0124] Example 1: Computer modeling

[0125] Figure 3A A structural model of SA(HaloTag)4 is shown, including a tetrameric streptavidin SA (center) and four bound biotinylated HaloTag protein molecules, each SA subunit corresponding to one HaloTag protein.

[0126] Figure 3BThe intrinsic mobility of the complex calculated from ANM analysis is shown. The vector length correlates with the amplitude of the protein domain motion. The HaloTag protein has an overall motion relative to the SA template. ANM is a computational tool for analyzing internal motions of molecular structures. Details of the computational methods used to build the structural model and calculate internal motions are described below.

[0127] All models were built using the 3D builder tool in Maestro software. The ionization state of the protein was assigned correctly according to the results of Schrodinger. MD simulations were performed using Desmond software. Optimized potential liquid simulation (OPLS)-3e force field was used in this system with a single-point charged (TIP3P) water model and 0.15 M NaCl to solvate the protein. An orthogonal water box was used to create a buffer zone between the protein atoms and the box sides. The temperature was kept constant at 310 K with a value of 2.0 fs in the integration step. We investigated the structure by performing 50 ns unbiased molecular dynamics (MD) analysis and exploited ANM calculations to analyze the protein backbone motion in the MD trajectory.

[0128] Example 2: Halotag PEG-biotin ligand synthesis

[0129] HaloTag-PEG4-biotin ligand was synthesized. The molecular formula of HaloTag- PEG4-biotin ligand is C 31 H 57 ClN4O9S, with a molecular weight of 697, and the chemical structure is as follows:

[0130]

[0131] The terminal -CI of HaloTag-PEG4-biotin ligand is used to be replaced by Asp106 of HaloTag protein in a nucleophilic reaction to form a stable ester bond (Los et al., 2008), thus forming biotinylated Halo-tag.

[0132] 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine hydrochloride and NHS-PEO4-biotin were used to synthesize

[0133] PEG-biotin ligand. The specific method is as follows; 2-(2-((6-chlorohexyl)oxy)ethoxy)ethylamine hydrochloride (85 μΐ, 2.55 x 10 -6 mol) was added to a stirred solution of NHS-PEO4-biotin (5 mg, 8.5 x 10 -5mol) to a 0.3 M solution of CH2Cl2, and finally a drop of excess diisopropylethylamine. The reaction mixture was stirred at room temperature for 4 hours, then diluted to 1 ml water and purified by preparative HPLC. H2O and acetonitrile were used as the solvent for the semi-preparative HPLC, and the absorbance of the reaction product was measured at 210 nm. The purity of the sample after purification was analyzed using LC-MS. 1% formic acid in water and acetonitrile were used as the solvent mobile phase.

[0134] The overall chemical reaction is as follows:

[0135]

[0136] Figure 4A Purified Halotag PEG-biotin ligand showing analysis by HPLC: the wavelength of absorption was 210 nm and the retention time was 10 minutes.

[0137] Figure 4B LC-MS of Halotag PEG-biotin ligand. MS: m / z calculated. For

[0138] C31H58ClN4O9S+: 697.36 (100%), 698.36 (36.6%), 699.36 (41%), 719.3 (12.13%), 720.3 (4.41%).

[0139] Example 3: Expression and purification of halotag protein

[0140] DNA fragments of wild-type halotag protein were amplified by PCR and cloned into the pET29a vector. For protein production, the plasmid was transformed into E. coli BL21(DE3) cells. Cells were grown at 37°C until OD600 = 0.6, then the sample was transferred to 18°C and protein was induced by the addition of 0.2 mM isopropyl β-D-l-thiogalactopyranoside (IPTG). After 18 hours of induction, cells were harvested by centrifugation and resuspended in lysis buffer containing 25 mM Tris-HCl, pH 7, 400 mM NaCl and 10 mM imidazole. The cell suspension was lysed by high pressure disruption (800 p.s.i.). The cell lysate was centrifuged at 12,000 x g for 20 minutes. The pellet was discarded and the supernatant was applied to a nickel affinity column equilibrated with binding buffer consisting of 25 mM Tris-HCl, pH 7.5, 400 mM NaCl and 10 mM imidazole. The protein bound to the column resin was first washed with 50 mM imidazole and then eluted with 250 mM imidazole. The obtained protein preparation was dialyzed twice against 25 mM Tris-HCl, pH 8.0, 100 mM NaCl. Finally, the halotag protein in monomeric form was isolated using size exclusion chromatography.

[0141] Example 4: Size exclusion chromatography of streptavidin-halotag protein complex

[0142] Streptavidin (purchased from IBA, Gottingen, Germany) was mixed with halotag PEG-biotin ligand in a 1 :4 molar ratio. After 3 hours of incubation, the halotag monomeric protein was added to the mixture. After overnight incubation, the halotag-PEG4-biotin ligand and the halotag protein were injected into a size exclusion chromatography column, and the formed streptavidin-halotag protein complex was separated from the reaction mixture.

[0143] Figure 5A Size exclusion chromatography of streptavidin-halotag protein complex is shown. Figure 5B SDS-PAGE of streptavidin tetramer (shown as streptavidin), halotag protein (shown as halotag), size exclusion chromatography fraction 1 and fraction 2 Figure 5A ). Size exclusion chromatography and SDS-PAGE show that a streptavidin-halotag protein complex was formed.

[0144] Example 5: Negative staining electron microscopy of streptavidin-halotag protein complex

[0145] Electron microscopy of negatively stained samples was used to assess protein quality. Briefly, 3 L of freshly purified streptavidin-halotag complex (0.025 mg / ml) was applied to copper grids supported by thin layer glow discharge carbon film (Nanoscience Instruments, Inc.) for 1 min. After adsorption, negative staining was performed with uranyl acetate (2% w / v) at room temperature. Negatively stained grids were examined using a FEI Talos L120C operated at 120 kV.

[0146] Figure 6 Representative electron micrographs of negatively stained preparations of streptavidin-halotag protein complex separated by size exclusion chromatography (corresponding to fraction 1 in FIG. 1) are shown. Figure 5A

[0147] Example 6: Grid preparation for Cryo-EM

[0148] Peak fractions collected from SEC were concentrated to 1 mg / mL and then centrifuged at 12,000 x g for 30 min at 4 °C. A total of 4 pL of sample was applied to glow discharge Quantifoil holey carbon grids (Quantifoil® Micro Tools, 2-2 pm / hole, 300 mesh) using a Vitrobot (FEI) for blotting with a blotting time of 2 seconds at 100% humidity and 4 °C, then frozen in liquid ethane cooled by liquid nitrogen. Frozen grids were carefully transferred and stored in liquid nitrogen until cryo-EM images were collected.

[0149] Example 7: Data collection and image processing for Cryo-EM

[0150] A total of 19060 movies were collected on a Titan Krios G3i (Thermofisher Scientific) operated at 300 kV, equipped with a Gatan Image Filter Continuum 1069 (operated with a slit width of 20 eV) and installed with a K3 Summit detector (Gatan, Inc.). EPU was used to automatically acquire micrographs in super-resolution counting mode with a nominal defocus range of -1.5 to -2.5 pm at a pixel size of 0.67 A. Each 32-frame movie was collected at a dose of 50 electrons per pixel per second over an exposure time of 2.1 seconds, resulting in a total dose on the sample of 10°, 20°, 30°, and 40° tilt data were collected to overcome preferred orientation issues of the streptavidin-halotag complex. All movie frames in each stack were aligned and dose weighted using MotionCor2, resulting in a pixel size of 0.67 A. 10°, 20°, 30°, and 40° tilt data were collected to overcome preferred orientation issues of the streptavidin-halotag complex. All movie frames in each stack were aligned and dose weighted using MotionCor2, resulting in a pixel size of 0.67 A. ​2x merged image of pixels. The patchctf module of the Cryosparc2 software was used to estimate the defocus value and the astigmatism parameters of the contrast transfer function (CTF). A total of 12655 micrographs were selected for further processing. To understand the subunit composition, a reference-free two-dimensional (2D) classification was performed. The 2D classification module of Cryosparc2 was used for further data processing. Initially, approximately 5850507 particles were picked from the selected micrographs and subjected to 2D classification. After performing 2D classification in Cryosparc, the best 2D class averages were selected by visual inspection for building the heterogeneous refinement ab initio reconstruction in cryoSPARC. After the heterogeneous refinement, a class showing intact features (particles) was selected and subjected to anisotropic refinement using C2 symmetry followed by local refinement to get the overall resolution of 3.6 A.

[0151] Figures 7A-7B Representative micrographs and 2D classification of the streptavidin-halotag complex are shown. Figure 7A are micrographs recorded using cryo-electron microscope operating at 300 kV, scale bar: 50 nm. Figure 7B is a representative 2D classification average image of the complex.

[0152] Figures 8A-8B Resolution estimation of the cryo-electron micrographs is shown. Specifically, Figure 8A shows the Fourier shell correlation (FSC) plot indicating the corrected resolution to be 3.6; Figure 8B depicts the 3D map of the streptavidin-halotag complex after anisotropic refinement.

[0153] Example 8: Expression and purification of NK1R-halotag fusion protein

[0154] The DNA coding sequence for the Halotag protein was inserted into the intracellular loop 3 of the NK1R coding sequence, then the construct was amplified by PCR and cloned into the pEG BacMam vector. HEK293F cells were cultured in SMM 293T-II media at 37°C in an incubated shaker at 8% CO2. The target protein was expressed by transient transfection of HEK293F cells using plasmid DNA encoding the NK1R-halotag fusion protein. Briefly, for a 1 liter HEK293F cell culture, 1 mg of plasmid DNA and 4 mg of 25-kDa linear polyethylenimine were pre-incubated in 25 mL of fresh media for 10 min, then the two media samples were mixed for a further 15 min incubation, then the mixture was added to the cells. Transfected cells were cultured for 72 hours before harvesting by centrifugation. For each batch of protein purification, 4 liters of transfected HEK293F cells were collected by centrifugation at 2000 x g. The cell pellet was resuspended in lysis buffer containing 20 mM HEPES (pH 7.5), 500 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.1 mM PMSF, and 1X protease inhibitors, and disrupted with a French press. The cell membranes were solubilized in lysis buffer containing 1% (w / v) N-dodecyl- -D-maltoside (DDM), 0.2% cholesteryl hemisuccinate trisalt (CHS), and 0.5 mM naltrexone, stirred at 4°C for 12 h. The solubilized NK1R-halotag protein was separated from the insoluble fraction by centrifugation at 40,000 x g for 1 h, and incubated with 2 ml of TALON metal affinity resin at 4°C for 3 h. The resin was then washed with 10 column volumes of buffer containing 20 mM Tris pH 7.5, 150 mM NaCl, 0.5 mM naltrexone, 0.1 mM PMSF, and 0.1% DDM, 0.02% CHS, and 30 mM imidazole. The resin was eluted with 5 column volumes of buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5 mM naltrexone, 0.1 mM PMSF, and 0.1% DDM, 0.02% CHS, and 250 mM imidazole. The eluted protein was collected and concentrated using a 100 kDa concentrator. Streptavidin (purchased from IBA, Gottingen, Germany) was conjugated to

[0155] Halotag-PEG4-biotin ligands were mixed at a 1 :4 molar ratio, four Halotag-PEG4-biotin ligands were bound to one streptavidin tetramer, named SA(Halotag PEG-Biotin ligand)n(n=l-4). After 3 hours of incubation, the NK1R-Halotag fusion protein was added to the mixture to covalently bind to SA(Halotag PEG-Biotin ligand)n halotag ligand. After overnight incubation, the reaction mixture was applied to size exclusion chromatography to isolate the formed streptavidin-NK1R-halotag complex from the reaction mixture.

[0156] Figure 9A Size exclusion chromatography of the streptavidin-NK1R-halotag complex is shown. Figure 9B SDS-PAGE of the streptavidin NK1R-halotag complex, NK1R-halotag, size exclusion chromatography fractions 1 to 4 are shown. Figure 9A ) Volume exclusion chromatography and SDS-PAGE indicated that the streptavidin NK1R-halotag complex was formed.

[0157] Unless otherwise indicated, the terms “comprises,” “has,” “includes” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”). Although specific embodiments of the application have been shown and described herein, it is understood that changes and modifications can be made to the specific embodiments without departing from the broader aspects of the application. Accordingly, the purpose of the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the application.

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Claims

1. A method for protein structure determination using cryo-electron microscopy, comprising: incorporating a tag into a target protein; binding the target protein comprising the tag to a scaffold protein to form a complex of the target protein and the scaffold protein; and performing single particle imaging using a cryo-electron microscope to determine the structure of the target protein complexed with the scaffold protein; wherein: the scaffold protein is any one of streptavidin, avidin, or a derivative thereof; the tag is used to selectively bind to the scaffold protein; when the target protein is a G-protein coupled receptor, GPCR, it is inserted with a biotin tag or biotinylated protein or polypeptide tag at one of the N-terminus, C-terminus, or one of the extracellular loops, or one of the intracellular loops, or it is selectively linked with an anticalin or an Ig-type or single chain antibody at one of the N-terminus, C-terminus, or one of the extracellular loops, or one of the intracellular loops; and when the target protein is a complex of a prototypical GPCR and an intracellular signaling protein, the tag is introduced to bind the target protein to the streptavidin or a derivative thereof as the scaffold protein by: 1) inserting a biotin tag, biotinylated protein or polypeptide tag at one of the N-terminus, C-terminus, or one of the extracellular loops, or one of the intracellular loops of the prototypical GPCR; 2) selectively linking an anticalin or an Ig-type or single chain antibody at one of the N-terminus, C-terminus, or one of the extracellular loops, or one of the intracellular loops of the prototypical GPCR; 3) fusing a biotin tag or biotinylated protein or polypeptide tag into the sequence of the intracellular signaling protein; 4) selectively binding a biotinylated antibody to the intracellular signaling protein; or 5) selectively binding a biotinylated anticalin to the intracellular signaling protein.

2. The method of claim 1, wherein, in the case that the tag is the biotin tag, the step of incorporating a tag into a target protein comprises: incorporating the biotin tag in the side chain of a non-canonical amino acid and site-specifically introducing the non-canonical amino acid into the amino acid sequence of the target protein; or chemically linking the biotin tag to the side chain of an amino acid and site-specifically introducing the amino acid into the target protein; or chemically linking the biotin tag to a glycosylation site of the N-terminal portion of the target protein.

3. The method of claim 1, wherein: the biotinylated protein or polypeptide tag is selected from one of the group consisting of: a self-labeling protein tag that allows site-specific covalent attachment of a biotin residue to the corresponding tag protein; an acyl carrier protein tag, ACP-tag, or a peptidyl carrier protein tag, PCP-tag; and Escherichia coli the self-labeling protein tag is a Snap-tag, Clip-tag, or Halo-tag. Avi-tag, which is suitable for fusion with the N- or C-terminus or an exposed loop region of the target protein and can be covalently linked to biotin by ​ the biotinylation enzyme BirA.

4. The method of claim 3, wherein, ​ 5. The method of claim 1, wherein, The target protein has a molecular weight between 20 kDa and 50 kDa.

6. The method of claim 1, wherein, The target protein is a water-soluble protein or a membrane protein.

7. The method of claim 1, wherein, The biotin tag, the biotinylated protein or polypeptide tag or Strep-tag is fused at the intracellular loop structure I4 of the GPCR, thereby stabilizing the bound streptavidin or streptactin in a rigid structure.

8. The method of claim 1, wherein, The biotin tag, the biotinylated protein or polypeptide tag or Strep-tag is fused at one of the extracellular loops of the GPCR, thereby stabilizing the bound streptavidin or streptactin in a rigid structure.

9. The method of claim 1, wherein, The intracellular signaling protein is selected from any one of the following: a heterotrimeric G protein or a mini G protein adapted to bind to the GPCR activated by an agonist; a G protein-coupled receptor kinase, abbreviated as GRK, adapted to bind to and phosphorylate the active GPCR; an arrestin adapted to bind to the phosphorylated GPCR; and a peptide sequence mimicking the receptor-binding region of a G protein or an arrestin.

10. The method of claim 1, wherein, When the target protein is the neurokinin 1 receptor, abbreviated as NK1R, the tag is a biotinylated Halo-tag, and the scaffold protein is streptavidin, a tetrameric NK1R is assembled on the streptavidin via the biotinylated Halo-tag, i.e. a complex SA(HaloTag-NK1R) is formed n wherein n is an integer between 1 and 4.

11. The method of claim 10, wherein, The biotinylated Halo-tag is inserted in the third intracellular loop structure IL3 of the NK1R and the amino acid sequence from position 227 to 237 is deleted.

12. The method of claim 10, wherein, Before the step of incorporating a tag into the target protein, the HaloTag-PEG4-biotin ligand is synthesized, and a stable ester bond is formed between the HaloTag-PEG4-biotin ligand and the HaloTag protein to form the biotinylated Halo-Tag tag.

13. The method of claim 12, wherein, The molecular formula of the HaloTag-PEG4-Biotin ligand is C 31 H 57 ClN4O9S, with a molecular weight of 697, which has the following chemical structure: Formula (I); wherein the terminal -Cl of the HaloTag-PEG4-biotin ligand is replaced by the Asp106 of the HaloTag protein in an affinity reaction to form a stable ester bond, thereby forming the biotinylated Halo-tag tag.

14. The method of claim 1, wherein, Before the step of incorporating a tag into the target protein, the method comprises molecular modeling for finding and adjusting the appropriate insertion position of the biotin tag or the biotinylated protein or polypeptide tag or Strep-tag in the sequence of the target protein to obtain the optimal structural rigidity between the target protein and the scaffold protein.

15. The method of claim 14, wherein, During the molecular modeling, additional spacer amino acids are inserted into the amino acid sequence of the target protein, or functionally irrelevant flexible amino acid sequences are removed from the amino acid sequence of the target protein.

16. The method of claim 15, wherein, The PEG4 spacer in the HaloTag-PEG4-biotin ligand is further shortened or enlarged to obtain the optimal structural rigidity between the scaffold protein and the target protein.

Images