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Jul 2020

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A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs
活性电泳分析测定G蛋白与GPCR偶联的生化表征   

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Abstract

G protein-coupled receptors (GPCRs) are a large family of membrane-embedded receptors that have diverse roles in physiology and are major drug targets. GPCRs transduce an agonist binding signal across the membrane to activate intracellular heterotrimeric G proteins. The dynamic nature of the receptors and the complexity of their interactions with agonists and G proteins present significant challenges for biochemical studies. Most biochemical/biophysical methods that have been employed to study GPCR-G protein coupling require purified receptors and are technically difficult. Here, we provide a protocol for a relatively simple and time- and cost-effective membrane protein native PAGE assay, to visualize and biochemically characterize agonist-dependent coupling of detergent-solubilized GPCRs to purified G protein surrogate “mini-G” proteins, which stabilize the receptor in an active state. The assay was developed for our studies of the calcitonin receptor-like receptor, a class B GPCR that mediates the actions of calcitonin gene-related peptide and adrenomedullin peptide agonists. It does not require a purified receptor and it can be used in a screening format with transiently-transfected adherent mammalian cell cultures, to quickly identify detergent-stable complexes amenable to study, or in a quantitative format with membrane preparations, to determine apparent affinities of agonists for the mini-G-coupled receptor and apparent affinities of mini-G proteins for the agonist-occupied receptor. The latter provides a partial measure of agonist efficacy. The method should be applicable to other GPCRs, and has the potential to be adapted to the study of other challenging membrane proteins and their complexes with binding partners.


Graphic abstract:

Visualizing agonist-dependent mini-G protein coupling and determining apparent binding affinities using the native PAGE assay quantitative formats.


Keywords: Mini-G (Mini-G), Membrane proteins (膜蛋白), G protein-coupled receptors (G蛋白偶联受体), Native PAGE (活性电泳), Peptide agonist (肽激动剂), Thermostability (热稳定性), Detergent solubilization (去污剂溶解), hrCNE (hrCNE)

Background

G protein-coupled receptors are a family of ~800 membrane-embedded receptors with critical physiologic roles in virtually every system of the human body and they are targeted by approximately one-third of FDA-approved therapeutics (Pierce et al., 2002; Sriram and Insel, 2018). Significant advances in biochemical, biophysical, and structural methods for the study of GPCRs have drastically improved our understanding of how an agonist-bound GPCR acts as a guanine nucleotide exchange factor, to activate intracellular heterotrimeric G proteins. Nonetheless, biochemical investigations of receptor-G protein coupling remain challenging due to the dynamic nature of the receptors and the complexity of their interactions with heterotrimeric G proteins, which are regulated by agonist binding to the GPCR, and guanine nucleotide binding to the G protein alpha subunit. Biochemical and biophysical methods that have been used to investigate this interaction include fluorescence- and nuclear magnetic resonance (NMR)-based methods (Yao et al., 2009; Gregorio et al., 2017; Huang et al., 2021). These methods are very powerful, but they are technically challenging and costly and require purified receptors in detergents or nanodiscs.


An alternative to working with G protein heterotrimers for biochemical studies is the use of “mini-G” proteins that were first developed as tools for GPCR structural studies (Carpenter and Tate, 2016; Nehmé et al., 2017). Mini-G proteins are minimal G protein alpha subunits that have enhanced stability in detergents and contain mutations that uncouple receptor binding from guanine nucleotide exchange, such that they trap GPCRs in an active state conformation, equivalent to that observed in structures of agonist-bound GPCRs in complex with nucleotide-free heterotrimer. Mini-G proteins are available for each of the four families of G protein alpha subunits. Biochemical assays for assessing mini-G interactions with GPCRs have been reported (Nehmé et al., 2017), including a fluorescent saturation binding assay using immobilized receptor, and an assay based on the fluorescence-detection size-exclusion chromatography (FSEC) technique (Kawate and Gouaux, 2006). These assays work with unpurified receptors in detergent-solubilized lysates and use GFP-tagged mini-G for detection.


We recently reported a novel biochemical assay for GPCR-mini-G coupling based on the high resolution clear native electrophoresis (hrCNE) technique, which is a membrane protein native-PAGE method compatible with fluorescently-labeled proteins (Wittig et al., 2007). We developed the assay during the course of our work to understand how calcitonin gene-related peptide and adrenomedullin peptide agonists and RAMP accessory proteins control G protein coupling of the class B GPCR calcitonin receptor-like receptor (CLR) (Roehrkasse et al., 2020). The method enables both rapid, cost-effective screening for detergent-stable GPCR-G protein complexes and quantitative studies of agonist-dependent receptor-mini-G coupling. It does not require a purified receptor. Instead, it uses an EGFP-tagged receptor transiently over-expressed in the mammalian HEK293S GnT1; cell line. In the screening format, adherent cell cultures are directly solubilized with detergent, subjected to a simple centrifugation step, and the supernatants are analyzed by native-PAGE with visualization of the receptor by in-gel fluorescence imaging. Exogenous addition of agonist peptides and purified mini-G yields a mobility shift indicative of complex formation. In the quantitative assay format, crude membrane preparations are used to improve reproducibility and increase throughput. In the first quantitative format, the agonist is varied in the presence of a constant excess of mini-G to generate a binding curve that measures the apparent affinity of the agonist for the mini-G-coupled receptor. In the second quantitative format, mini-G is varied in the presence of a constant receptor-saturating concentration of agonist to generate a binding curve that measures the apparent affinity of mini-G for the agonist-occupied receptor. This second format provides a partial measure of agonist efficacy (Roehrkasse et al., 2020).


The native PAGE method proved to be a relatively simple, inexpensive, and highly versatile assay, which allowed us to investigate the biochemistry of agonist and G protein interactions with CLR:RAMP complexes. The screening and quantitative assay formats are described in detail here. We have also found the method to be very useful in a thermostability format, which is not described here, but can be found in our original report (Roehrkasse et al., 2020). This assay should be applicable to other GPCRs and may be valuable for the biochemical study of other challenging membrane proteins and their binding partners.

Materials and Reagents

  1. Plasticware

    15 ml conical tubes (VWR, catalog number: 76176-950)

    48-well cell culture plate (Corning, Costar, catalog number: 3548)

    Microfuge tubes (VWR, catalog number: 87003-294)

    T-75 culture flask (Corning, catalog number: 43064)

  2. Cell lines

    HEK293S GnT1 (ATCC, catalog number: CRL-3022) (see Note 3)

  3. Cell culture reagents

    DMEM with 4.5 g/L Glucose and L-Glutamine (Lonza, catalog number: 12-604Q)

    Fetal Bovine Serum (FBS) (Gibco, catalog number: 16000-044)

    Non-Essential Amino Acids (NEAA) (Gibco, catalog number: 11140-050)

    Penicillin/Streptomcyin (Gibco, catalog number: 15140-122)

    Phosphate Buffered Saline (PBS) (Gibco, catalog number: 10010-023)

    Polyethylenimine, branched (PEI) (Sigma-Aldrich, catalog number: 408727-100 ml)

  4. Salts – stored at room temperature

    Ammonium persulfate (APS) (Bio-Rad, catalog number: 1610700)

    Calcium Chloride (CaCl2) (Sigma-Aldrich, catalog number: 223506-500G)

    HEPES Sodium Salt (Sigma-Aldrich, catalog number: H7006-500G)

    Potassium Chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G)

    Magnesium Chloride (MgCl2) (Sigma-Aldrich, catalog number: 442611-500G)

    Sodium Chloride (NaCl) (EMD Millipore, catalog number: SX0420-5)

    Valproic acid sodium salt (Sigma-Aldrich, catalog number: P4543-25G)

  5. Alcohols

    Isoproponol (VWR, BDH, catalog number: BDH1133-4LG)

    Methanol (Supelco, EMD Millipore, catalog number: MX0475-1)

  6. Solubilizing chemicals – stored at -20°C

    Lauryl Maltose Neopentyl Glycol (LMNG) (Anatrace, catalog number: NG310-25GM)

    Cholesteryl Hemisuccinate (CHS) (Anatrace, catalog number: CH210-5GM)

  7. Other Chemicals

    6-amino hexanoic acid (Sigma-Aldrich, catalog number: 07260-1KG)

    30% Acrylamide/Bis-acrylamide 29:1 (Acryl/Bis) (Bio-Rad, catalog number: 1610156)

    Fatty-Acid-Free BSA (FAF BSA) (PAA Laboratories, catalog number: K35-002)

    Glutathione oxidized (GSSG) (Sigma-Aldrich, catalog number: G4376-10G)

    Glutathione reduced (GSH) (Sigma-Aldrich, catalog number: G4251-25G)

    Imidazole (EMD Millipore, catalog number: 5720-500GM)

    Sodium Hydroxide (NaOH) (VWR, BDH, catalog number: BDH3247-1)

    Tetramethylenediamine (TEMED) (Bio-Rad, catalog number: 161-0800)

    Tricine (Sigma-Aldrich, catalog number: T0377-250G)

    Protease Inhibitor tablets (Thermo Scientific, Pierce, catalog number: A32955)

  8. Peptides

    Human α calcitonin gene-related peptide (Bachem, catalog number: 4013281)

    Human adrenomedullin (Bachem, catalog number: 4034489)

    Human adrenomedullin2/intermedin (Bachem, catalog number: 4044529)

  9. Enzymes

    Apyrase (New England BioLabs, catalog number: M0398L)

  10. Membrane preparations and purified proteins

    Crude membrane preparation from HEK293S GnT1 cells transiently co-expressing tagged MBP-CLR-EGFP and MBP-RAMP, using the pHLsec expression vector (Aricescu et al., 2006); the preparation method was published in Roehrkasse et al. (2020) – flash-frozen in liquid nitrogen and stored at -80°C. The final preparationss are stored in 25 mM HEPES, pH 7.5, 10% (v/v) glycerol, 25 mM NaCl, 2 mM MgCl2, and 1× protease inhibitor tablet. Protein concentrations are ~4-6 mg/ml with the receptor at ~100 nM.

    Purified H6-SUMO-mini-G proteins (mini-Gs, mini-Gs/i, and mini-Gs/q were used; the present protocol utilizes example data from miniGs); methods published in Carpenter and Tate (2016 and 2017), Nehmé et al. (2017) , and Roehrkasse et al. (2020) – stored at -80°C. The purified proteins are stored at ~25-30 mg/ml in a buffer of 25 mM HEPES, pH 7.5, 50% (v/v) glycerol, 150 mM NaCl, 0.5 mM DTT, 1 mM MgCl2, and 1 μM GDP (see Notes 1 and 2).

  11. High resolution clear native PAGE gel (see Recipes)

  12. 4× Binding Buffer (see Recipes)

  13. 4× Detergent Buffer (see Recipes)

  14. Running Buffers (see Recipes)

  15. Culture media (see Recipes)

  16. Transfection media (see Recipes)

  17. 100 mM GSH and GSSG redox buffer stocks (see Recipes)

Equipment

  1. Mini-PROTEAN Electrophoresis apparatus (Bio-Rad, model: 1658001FC)

  2. Chemidoc MP imager (Bio-Rad, model: 12003154)

  3. CO2 incubator (NuAire, model: NU-5800)

  4. Imager tray (Bio-Rad, catalog number: 12003028)

  5. Rocking shaker (Reliable Scientific, model: 55)

  6. Tube tumbler (Labnet International, model: H5500)

  7. Benchtop Micro centrifuge (Eppendorf, model: 5415R)

Software

  1. ImageLab (Bio-Rad, https://tinyurl.com/2v4t44re)

  2. Prism (GraphPad, https://www.graphpad.com/)

Procedure

  1. Preparation: High resolution clear native PAGE (hrCNE) gels (Wittig et al., 2007) and buffer stocks

    1. Prepare hrCNE gels according to the recipe section below (see Recipe 1, Table 2). We used an 8% resolving gel, but other resolving gel percentages could be used (see Note 12).

    2. Prepare 4× binding buffer and 4× solubilization buffer stocks according to the recipes below (Recipes 2 and 3).

    3. Prepare the hrCNE 20× Cathode and 50× Anode buffers and store them at room temperature in a dark cabinet (Recipe 4).


  2. Adherent cell culture – For adherent cell culture screening format (see Note 4)

    1. Preparation: Cell media

      1. Culture media (see Recipe 5).

      2. Transfection media (see Recipe 6).

    2. Seeding

      1. Seed 120,000 cells/well of HEK293S GnT1– cells with 250 μl culture media in a cell culture-treated 48-well clear plate.

      2. Incubate the plate at 37°C with 5% CO2 for 24 h and grow to ~90% confluency.

    3. Transient transfection of DNA using PEI

      Prepare plasmid DNA transfection mixtures and add to cells.

      1. Ingredients

        1). DMEM (remaining volume for a total of 30 μl).

        2). Receptor expression plasmid DNA (300 ng total per well) (see Note 5).

        3). PEI for a 1.5:1 ratio to DNA (0.45 μl of 1 mg/ml stock).

        4). 46.67 mM valproic acid (2.8 μl of 500 mM stock).

      2. Assembly and addition to cells

        1. For the transfection mix, combine ingredients numbered 1)-3) to microfuge tubes (order of addition DMEM  DNA  PEI) and mix well by pipetting up and down.

        2. Incubate for 10 min at room temperature.

        3. While the transfection mixture is incubating, carefully aspirate media from the 48-well plate and replace with 250 μl of transfection media.

        4. Add valproic acid (ingredient number 4) to microfuge tubes and mix well.

        5. Transfer 30 μl of transfection mixture to the appropriate well in the 48-well plate, and mix by gently swirling plate.

        6. Incubate plate at 30°C with 5% CO2 for 72 h.


  3. Preparation: Day of the experiment (screening format or quantitative format)

    1. Prepare the 1× Cathode and 1× Anode buffers by diluting the 20× and 50× stocks in ddH2O and chill at 4°C for several hours prior to running the native gel, add detergent system to the Cathode buffer.

      1. 1× Cathode buffer (see Recipe 4).

      2. 1× Anode buffer (see Recipe 4).

    2. Thaw 4× binding buffer and 4× detergent buffer stocks, apyrase, and peptide agonists on ice.

    3. Make 1× binding buffer with 0.1 mg/ml FAF-BSA: For 500 μl total volume, combine 372.5 μl of ddH2O with 125 μl 4× binding buffer and 2.5 μl 20 mg/ml FAF-BSA (dissolved in ddH2O).

    4. Dilute 500 U/ml apyrase stock

      1. Screening Format: 1:10 in binding buffer with FAF-BSA to 50 U/ml and keep on ice.

      2. Quantitative Formats: 1:100 in binding buffer with FAF-BSA to 5 U/ml and keep on ice.

    5. Make 4× detergent buffer with 0.2 U/ml apyrase: For 200 µl total volume, combine 199.2 µl 4× detergent buffer with 0.8 µl 50 U/ml apyrase dilution (see Note 6).

    6. Thaw membrane prep and mini-G protein on ice right before setting up the assay.

    7. Make 100 mM GSH and GSSG redox buffer stocks (see Recipe 7) (see Note 7):

    8. Use the 100 mM GSH and 100 mM GSSG stocks to make working stocks for a specific assay format.

      1. Screening Format

        2.4 mM GSH/0.48 mM GSSG stock (for 100 μl: 97.12 μl ddH2O, 2.4 μl of 100 mM GSH, 0.48 μl of 100 mM GSSG).

      2. Quantitative Formats

        50 mM GSH/10 mM GSSG stock (for 100 μl: 40 μl ddH2O, 10 μl of 100 mM GSSG, 50 μl of 100 mM GSH).


  4. Adherent cell culture screening format

    1. Binding Reaction

      The final reaction is composed of 25 μl 4× detergent buffer and 75 μl binding reaction by volume; the binding reaction is assembled in microfuge tubes prior to introduction to the transfected cells in the 48-well plate.

      1. Ingredients (in order of addition)

        1). 1× Binding buffer (remaining volume for a total of 75 μl).

        2). 200 μM GSH/40 μM GSSG (6.25 μl of 2.4 mM GSH/0.48 mM GSSG stock).

        3). 4% v/v Glycerol (6 μl from 50% v/v stock).

        4). 66.66 μM mini-G protein (the volume depends on mini-G stock concentration).

        5). 13.33 μM peptide agonist (the volume depends on peptide stock concentration).

      2. Assembly and addition to cells

        1. Combine ingredients numbered 1)-4) and mix well.

        2. Incubate for 30 min on ice, to allow for reduction of the mini-G protein.

        3. Add the peptide agonist (ingredient number 5) to the desired reactions and mix well.

        4. Aspirate media from cells in the 48-well plate.

        5. Wash wells with 250 μl 1× PBS.

        6. Aspirate PBS and place plate on ice.

        7. Add 75 μl of binding reaction to each well of cells, swirl plate to mix.

        8. Incubate the 48-well plate on ice for 30 min.

    2. Solubilization

      1. Add 25 μl of 4× Detergent buffer ± 0.2 U/ml apyrase to each well and swirl plate to mix (see Note 6).

      2. Place the plate on a rocker at 4°C and allow cells to solubilize for 2 h.

      3. Pipette lysates to pre-chilled microfuge tubes on ice.

      4. Analyze by hrCNE gel electrophoresis and image gel as in Procedure F-G.


  5. Quantitative Assay Formats

    1. Quantitative assay binding format 1 (Figure 1) – agonist apparent affinity for the mini-G-coupled receptor

      1. The final reaction is composed of ¼ 4× detergent buffer and ¾ binding reaction by volume; the binding reaction is assembled from a mastermix and agonist serial dilutions, to produce the final desired concentrations (see Table 1 for concentrations at each step).

      2. To maximize reproducibility, combine reaction components that will be equal in all reactions in a mastermix (MM) (receptor membrane prep, an excess of the mini-G protein, and apyrase); GSH and GSSG are added with the mini-G to keep the mini-G protein reduced (see Note 7).

      3. Mastermix:

        1. Ingredients (in order of addition with volumes for 400 μl MM shown)

          1). ddH2O (remaining Volume for a total of 400 μl).

          2). 1× Binding buffer (100 μl of 4× stock).

          3). 0.1 mg/ml FAF-BSA (2 μl of 20 mg/ml).

          4). 400 μM GSH/80 μM GSSG (3.2 μl of 50 mM GSH/10 mM GSSG stock).

          5). 0.133 U/ml apyrase (10.64 μl of 5 U/ml stock) (see Note 8).

          6). 133.5 μM mini-G protein (the volume depends on mini-G stock concentration) (see Note 9).

          7). 26.67 nM receptor membrane prep (106.68 μl of 100 nM stock).

        2. Assembly

          Combine ingredients numbered 1)-6) and mix well.

          Pre-incubate for 30 min on ice, to allow for reduction of the mini-G protein.

          Add the receptor membrane prep and mix well by pipetting up and down.

      4. Agonist dilutions:

        1. Dilute the agonist peptide to 2.67× of the final desired highest concentration in 1× binding buffer with 0.1 mg/ml FAF-BSA (see Table 1 for concentrations) in microfuge tubes on ice.

        2. Serially dilute the agonist peptide 3-fold in 1× binding buffer with 0.1 mg/ml FAF-BSA (e.g., dilute 10 μl of the agonist dilution in 20 μl of 1× binding buffer with 0.1 mg/ml FAF-BSA).

      5. Binding reaction: Assemble the binding reaction by combining equal volumes (15 μl + 15 μl) of the agonist dilution and mastermix.

      6. Incubate binding reaction on ice for 30 min and proceed to solubilization (Step E3).

    2. Quantitative assay binding format 2 – mini-G apparent affinity for the agonist-occupied receptor

      1. The final reaction is composed of ¼ 4× detergent buffer and ¾ binding reaction, as in assay format 1; the binding reaction is assembled from a mastermix and miniG serial dilutions to produce the final desired concentrations (see Table 1).

      2. To maximize reproducibility, combine reaction components that will be equal in all reactions in a mastermix (MM) [10 nM of the receptor complex, an excess (10 μM) of the agonist (see Note 14)]; GSH and GSSG are added with the mini-G in the serial dilution, to keep the mini-G protein reduced.

      3. Mastermix:

        1. Ingredients (in order of addition with volumes for a 400 μl MM shown)

          1). ddH2O (remaining Volume for a total of 400 μl).

          2). 1× Binding buffer (100 μl of 4× stock).

          3). 0.1 mg/ml FAF-BSA (2 μl of 20 mg/ml).

          4). 0.133 U/ml apyrase (10.64 μl of 5 U/ml stock) (see Note 8).

          5). 26.67 μM agonist (the volume depends on agonist stock concentration).

          6). 26.67 nM receptor complex (106.68 μl of 100 nM stock).

        2. Assemble all ingredients in the order indicated and mix well.

      4. Mini-G dilutions:

        1. Dilute the mini-G protein to 2.67× of the final desired highest concentration (200 μM) in 1× binding buffer with 0.1 mg/ml FAF-BSA.

        2. Add a 3-fold molar excess of GSH/GSSG (600 μM), mix well, and pre-incubate for 30 min on ice, to reduce mini-G.

        3. Serially dilute the mini-G 3-fold in 1× binding buffer with 0.1 mg/ml FAF-BSA (e.g., dilute 10 μl of the agonist dilution in 20 μl of 1× binding buffer with 0.1 mg/ml FAF-BSA); note that the GSH/GSSG is diluted proportionally with the mini-G protein.

      5. Binding reaction: Assemble the binding reaction by combining equal volumes (15 μl + 15 μl) of the mini-G dilution and mastermix.

      6. Incubate binding reaction on ice for 30 min, and proceed to solubilization (Step E3).



      Figure 1. Quantitative Assay Formats Overview.

      Depiction of key steps of the quantitative hrCNE gel assay format corresponding to Procedure C-G and the Data analysis in the protocol.


      Table 1. Buffer and binding reaction components concentrations at various stages of the protocol.

      1Binding buffer: 25 mM HEPES, pH 7.5, 140 mM NaCl, 10 mM KCl, 1 mM MgCl2, 2 mM CaCl2 + Pierce Protease Inhibitor (PI).

      2GSH:GSSG is at 5:1 molar ratio throughout, and GSH:miniG ratio is at a 3:1 ratio throughout for mini-Gs and mini-Gs/q; for additional information about mini-Gs/i see Roehrkasse et al. (2020).


    3. Solubilization

      1. Mix the thawed 4x detergent buffer by pipetting up and down.

      2. Add 10 μl of 4× detergent buffer (Recipe 3) to each of the binding reactions; this will bring all concentrations to 1× of the final concentration

      3. Place the microfuge tubes on a tube tumbler in a cold room (4°C), and allow reactions to solubilize for 2 h (see Note 10).

      4. Final buffer conditions:

        25 mM HEPES, pH 7.5

        140 mM NaCl

        10 mM KCl

        1 mM MgCl2

        2 mM CaCl2

        0.5% w/v LMNG/0.05% w/v CHS

        Pierce Protease Inhibitor at 0.538×

        Note: This is from buffer stocks only, the final solution also includes components from the mini-G storage buffer, the amount added varies with assay format, as well as the membrane preparation storage buffer.The final concentrations of glycerol in our samples vary from 1% v/v in samples without added mini-G protein to 3% in samples with the highest amount of mini-G protein.


  6. Gel Electrophoresis (see Notes 11 and 12)

    1. Set up the hrCNE gel in a cold room (4°C) with the pre-chilled cathode and anode buffers prepared in Procedure C.

    2. Pre-run gel at 100 V for 20 min to allow the detergent to enter the gel.

    3. Place the 1.5 ml microfuge tubes from Steps D2 or E3 in a microfuge pre-chilled to 4°C.

    4. Spin at 16,100 × g for 10 min at 4°C in the microcentrifuge (Eppendorf 5415R).

    5. Load 20 μl of each reaction supernatant into the wells. This can be a challenging step and may take some practice because no loading dye is used.

    6. Run the gel at 200 V for 3.5 h.


  7. Image the gel by in-gel fluorescence

    1. Gently separate the gel from glass plates and rinse three times in ddH2O.

    2. Image with the BioRad ChemiDoc MP imager, using the blot/UV/stain-free imaging plate.

    3. Preset program: ProQ Emerald 488 for EGFP fluorescence.

    4. Exposure times vary depending on band intensity. For quantitative gels, the optimal exposure time was determined and all gels were imaged for the same time; 80-120 s is a reasonable starting point.


  8. Quantify band intensity using the Bio-Rad Image laboratory software

    1. Tutorial videos are available from Bio-Rad on YouTube: https://www.youtube.com/watch?v=IV_P47ScoYo&list=PLrAEgIY86I6zytNeMPGBhvXnKIm5MS-6J&index=3&t=8s.

    2. Use the Image tools to straighten/rotate the image as needed.

    3. Use the lane and band tool to define lanes and select bands

      1. Manually assign 10 lanes and adjust the lanes to align with the gel.

      2. Use the “add bands” function to select bands for all dimer and quaternary complex bands.

      3. In the area where the bands are not easily visible, you can look at the lane profile tool to see faint bands.

    4. Use the lane profile view to select/adjust the width of the band peaks.

      1. Background can be adjusted and excluded as needed, by using the background selection.

      2. Review the width for each band and adjust as needed. Pay attention to band widths, to ensure that they are uniform.

    5. Export data using the analyze data function.

Data analysis

Plot the adjusted volume determined by densitometry against the agonist (format 1) or mini-G (format 2) concentration on a log scale. Fit the agonist and mini-G binding data using a 3 or 4 parameter logistic equation by nonlinear regression, and determine a pEC50 (quaternary complex appearance), or pIC50 value (dimer disappearance), to determine the apparent binding affinities (see Note 13). Both the appearance of the quaternary complex band, and the disappearance of the dimer band can be plotted. We have seen good agreement between the pEC50 and pIC50 values, if the mini-G/agonist affinity is sufficiently high to fully visualize the transition from the dimer to the quaternary complex.

Notes

  1. We produced the mini-G proteins as SUMO-fusion proteins originally for purification purposes, but we found that the fusions worked better than free mini-G proteins for some receptor complexes, presumably because their larger size yielded a better mobility shift. Different forms of mini-G may need to be tested to find the best mobility shift.

  2. Purification of mini-G12 has been reported (Nehmé et al., 2017). We did not use this protein in our assays, but we assume it would work as well.

  3. The HEK293S GnT1 cell line lacks N-acetyl-glucosaminyltransferase I activity, and therefore produces homogenous N-glycans, which enables sharp defined bands in the gels.

  4. Use the appropriate sterile technique in a cell culture hood.

  5. We used CLR that was tagged at the N-terminus with maltose-binding protein (MBP) and at the C-terminus with EGFP. The RAMP subunit was also N-terminally tagged with MBP. MBP seemed to allow more defined bands on the gels, but this may not be necessary in all cases. In addition, the flexible receptor C-tail was truncated. The vector used for expression was the pHLsec vector previously described (Aricescu et al., 2006).

  6. 4x Detergent buffer + 0.2 U/ml apyrase was used in wells that contained mini-G. 4× Detergent buffer w/o apyrase was used in all other wells.

  7. The mini-G proteins have several solvent-exposed cysteine residues that have a tendency to form intermolecular disulfide bonds when stored at high concentrations. We used the GSH/GSSG redox buffer to reduce the mini-G, without damaging our receptors and peptide agonists, which contain several disulfide bonds. In some cases, simpler use of a low concentration of DTT or TCEP to reduce mini-G may be possible.

  8. In the original paper, we added apyrase as described for the quantitative formats, but we have since moved to adding the apyrase during the solubilization step, as described for the adherent screening format.

  9. We have used both 25 and 50 μM final mini-G concentrations.

  10. The 30 min binding reaction and 2 h solubilization were sufficient to reach equilibrium for most of the interactions we studied, but longer times may be needed in some cases. This should be tested on a case-by-case basis.

  11. It is beneficial to have designated equipment for native gels, to avoid possible carry-over of residual SDS from previous SDS-PAGE gel runs.

  12. The acrylamide percentage used for the hrCNE resolving gels should be optimized. For our purposes, the 8% resolving gel worked well. Gradient gels can also be considered.

  13. Interactions between agonists, GPCRs, and G proteins are complex and have allosteric effects in both directions. In addition, there is the possibility of perturbation of the equilibrium during electrophoresis. For these reasons, we describe the affinities derived from the quantitative formats as “apparent binding affinities”.

  14. Agonist concentration should be chosen to saturate the receptor (100× KD if possible).

Recipes

  1. High resolution clear native PAGE gel (see Table 2)

    1. Stock solution of 0.1 M Imidazole (pH 7.0)/2 M 6-amino hexanoic acid can be prepared in a larger volume (250 ml) and stored in the dark at room temperature.

    2. Selection of the resolving gel percentage will depend on the size of the protein complex to be visualized.

    3. At room temperature in a 15 ml conical tube, combine ddH2O, 0.1 M Imidazole (pH 7.0)/2 M 6-amino hexanoic acid, and 30% Acryl:Bis and mix well to combine.

    4. Add the APS and TEMED, mix well and immediately cast the resolving gel.

    5. Top the gel off with isopropanol and allow the gel to solidify.

    6. Pour off the isopropanol and gently wipe clean with a tissue.

    7. Assemble stacking gel as done with the resolving gel, cast with a 10 well comb, and allow the gel to solidify.

    8. Gels can be stored at 4°C for several days wrapped in moist paper towels surrounded by plastic wrap.


      Table 2. hrCNE gel recipe.

      Resolving Stacking
      % Acryl:Bis 8% 10% 12% 6%
      ddH2O 4.74 ml 4.06 ml 3.4 ml 2.29 ml
      0.1 M Imidazole (pH 7.0)/2 M 6-amino hexanoic acid 2.5 ml 2.5 ml 2.5 ml 1 ml
      30% Acryl:Bis 2.66 ml 3.34 ml 4 ml 0.67 ml
      10% APS 100µl 100 µl 100 µl 40 µl
      TEMED 4 µl 4 µl 4 µl 4 µl
      Total Volume 10 ml 10 ml 10 ml 4 ml


  2. 4× Binding Buffer

    100 mM HEPES, pH 7.5, 560 mM NaCl, 40 mM KCl, 4 mM MgCl2, 8 mM CaCl2 + 1× Pierce Protease Inhibitor (PI) – 10 ml stock


    Stock solutions

    1 M HEPES (pH 7.5) stock

    5 M NaCl

    1 M KCl

    1 M MgCl2

    1 M CaCl2

    Store HEPES at 4°C

    All others can be stored at room temperature.


    For a 10 ml stock solution

    1 ml of 1 M HEPES, pH 7.5

    1.12 ml of 5 M NaCl

    0.4 ml of 1 M KCl

    0.04 ml of 1 M MgCl2

    0.08 ml of 1 M CaCl2

    7.36 ml of ddH2O

    1 PierceTM Protease Inhibitor Mini Tablets (1× in 10 ml)


    Add all ingredients to a 15 ml conical tube with the Pierce protease inhibitor tablet and vortex to combine (the protease inhibitor tablets are difficult to dissolve).

    Store at -20°C in 1 ml aliquots.

  3. 4× Detergent Buffer

    25 mM HEPES, pH 7.5, 140 mM NaCl, 10 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 2% w/v LMNG/0.2% w/v CHS


    Stock solutions

    Same stock solutions as in Recipe 2; detergent stock: 10% w/v LMNG/1% w/v CHS in ddH2O, stored in 1 ml aliquots at -20°C.


    For a 10 ml stock solution

    0.25 ml of 1 M HEPES, pH 7.5

    0.28 ml of 5 M NaCl

    0.1 ml of 1 M KCl

    0.01 ml of 1 M MgCl2

    0.02 ml of 1 M CaCl2

    2 ml of 10% w/v LMNG/1% w/v CHS

    7.34 ml of ddH2O

    1 PierceTM Protease Inhibitor Mini Tablets (1× in 10 ml)


    Add all ingredients except detergent to a 15 ml conical tube with the Pierce protease inhibitor tablet and vortex to combine (the protease inhibitor tablets are difficult to dissolve).

    Add the detergent and gently, but thoroughly, mix to combine.

    Store at -20°C in 1 ml aliquots.

  4. Running Buffers

    1× Cathode buffer: 50 mM Tricine, 7.5 mM Imidazole, pH 7.0, 0.01% w/v LMNG/0.001% w/v CHS

    1× Anode buffer: 25 mM Imidazole, pH 7.0

    20× Cathode buffer stock: 1 M Tricine, 150 mM Imidazole, pH 7.0

    50× Anode buffer stock: 1.25 M Imidazole, pH 7.0

  5. Culture media

    DMEM

    10% FBS

    1× NEAA

  6. Transfection media

    DMEM

    2% FBS

    1× NEAA

    50 U/ml Penicillin

    50 μg/ml Streptomycin

  7. 100 mM GSH and GSSG redox buffer stocks (see Note 7)

    1. Weigh GSH and GSSG powder and add to separate 15 ml conical tubes.

    2. Add ddH2O to the final desired concentration and vortex to dissolve.

    3. Adjust the pH to neutral by the addition of NaOH.

Acknowledgments

This work was supported by grants NIH R01GM104251 and a Presbyterian Health Foundation seed grant (AAP), and NIH predoctoral MD/PhD fellowship 1F30 HL142232 (AMR). This method was originally described in Roehrkasse et al. (2020).

Competing interests

None of the authors have any competing interests to disclose.

Ethics

This protocol utilizes a standard immortalized human cell line and did not involve human or animal subjects.

References

  1. Aricescu, A. R., Lu, W. and Jones, E. Y. (2006). A time- and cost-efficient system for high-level protein production in mammalian cells. Acta Crystallogr D Biol Crystallogr 62(Pt 10): 1243-1250.
  2. Carpenter, B. and Tate, C. G. (2016). Engineering a minimal G protein to facilitate crystallisation of G protein-coupled receptors in their active conformation. Protein Eng Des Sel29(12): 583-594.
  3. Carpenter, B. and Tate, C. G. (2017). Expression and purification of MiniG proteins from Escherichia coli. Bio-protocol 7(8): e2235.
  4. Gregorio, G. G., Masureel, M., Hilger, D., Terry, D. S., Juette, M., Zhao, H., Zhou, Z., Perez-Aguilar, J. M., Hauge, M., Mathiasen, S., et al. (2017). Single-molecule analysis of ligand efficacy in beta2AR-G-protein activation. Nature 547(7661): 68-73.
  5. Huang, S. K., Pandey, A., Tran, D. P., Villanueva, N. L., Kitao, A., Sunahara, R. K., Sljoka, A. and Prosser, R. S. (2021). Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling. Cell 184(7): 1884-1894 e1814.
  6. Kawate, T. and Gouaux, E. (2006). Fluorescence-detection size-exclusion chromatography for precrystallization screening of integral membrane proteins. Structure 14(4): 673-681.
  7. Nehmé, R., Carpenter, B., Singhal, A., Strege, A., Edwards, P. C., White, C. F., Du, H., Grisshammer, R. and Tate, C. G. (2017). Mini-G proteins: Novel tools for studying GPCRs in their active conformation. PloS one 12(4): e0175642.
  8. Pierce, K. L., Premont, R. T. and Lefkowitz, R. J. (2002). Seven-transmembrane receptors. Nat Rev Mol Cell Biol 3(9): 639-650.
  9. Roehrkasse, A. M., Warner, M. L. Booe, J. M., and Pioszak, A. A. (2020). Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay. J Biol Chem 295(28): 9736-9751.
  10. Sriram, K. and Insel, P. A. (2018). G Protein-Coupled Receptors as Targets for Approved Drugs: How Many Targets and How Many Drugs? Mol Pharmacol 93(4): 251-258.
  11. Wittig, I., Karas, M. and Schagger, H. (2007). High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6(7): 1215-1225.
  12. Yao, X. J., Velez Ruiz, G., Whorton, M. R., Rasmussen, S. G., DeVree, B. T., Deupi, X., Sunahara, R. K. and Kobilka, B. (2009). The effect of ligand efficacy on the formation and stability of a GPCR-G protein complex. Proc Natl Acad Sci U S A 106(23): 9501-9506.


简介

[摘要] G蛋白偶联受体(GPCRs)是一大类膜嵌入受体,在生理学中具有多种作用,是主要的药物靶点。GPCR 跨膜转导激动剂结合信号以激活细胞内异源三聚体 G 蛋白。受体的动态特性及其与激动剂和 G 蛋白相互作用的复杂性对生化研究提出了重大挑战。大多数用于研究 GPCR-G 蛋白偶联的生化/生物物理方法都需要纯化的受体,并且在技术上很困难. 在这里,我们提供了一个相对简单、时间和成本效益的膜蛋白天然 PAGE 测定的协议,以可视化和生化表征洗涤剂溶解的 GPCR 与纯化 G 蛋白替代“迷你 G”蛋白的激动剂依赖性偶联,其中使受体稳定在活性状态。该测定是为我们研究降钙素受体样受体而开发的,该受体是一种 B 类 GPCR,可介导降钙素基因相关肽和肾上腺髓质素肽激动剂的作用。它不需要纯化的受体,可用于瞬时转染贴壁哺乳动物细胞培养物的筛选形式,以快速鉴定适合研究的洗涤剂稳定复合物,或以膜制剂的定量形式,确定表观亲和力mini-G 偶联受体的激动剂和 mini-G 蛋白对激动剂占据的受体的表观亲和力。后者提供了激动剂功效的部分测量。该方法应适用于其他 GPCR,并有可能适用于其他具有挑战性的膜蛋白及其与结合伙伴的复合物的研究。

图文摘要:

使用天然 PAGE 测定定量格式可视化依赖激动剂的 mini-G 蛋白偶联并确定表观结合亲和力。


[背景] G 蛋白偶联受体是一个由约 800 种膜嵌入受体组成的家族,几乎在人体的每个系统中都具有关键的生理作用,大约三分之一的 FDA 批准的疗法将它们作为目标(Pierce等人, 2002 年;斯里拉姆和因塞尔,2018 年)。用于 GPCR 研究的生物化学、生物物理和结构方法的重大进展极大地提高了我们对激动剂结合的 GPCR 如何作为鸟嘌呤核苷酸交换因子激活细胞内异源三聚体 G 蛋白的理解。尽管如此,由于受体的动态特性及其与异源三聚体 G 蛋白相互作用的复杂性,受体-G 蛋白偶联的生化研究仍然具有挑战性,异源三聚体 G 蛋白受激动剂结合 GPCR 和鸟嘌呤核苷酸结合 G 蛋白 α 的调节亚基。已用于研究这种相互作用的生化和生物物理方法包括基于荧光和核磁共振 (NMR) 的方法(Yao等人,2009 年;Gregorio等人,2017 年;Huang等人,2021 年)。这些方法非常强大,但它们在技术上具有挑战性且成本高昂,并且需要清洁剂或纳米圆盘中的纯化受体。
使用 G 蛋白异源三聚体进行生化研究的另一种方法是使用“mini-G”蛋白,该蛋白最初被开发为 GPCR 结构研究的工具(Carpenter 和 Tate,2016 年;Nehmé等人,2017 年)。Mini-G 蛋白是最小的 G 蛋白 α 亚基,其在去垢剂中具有增强的稳定性,并且包含将受体结合与鸟嘌呤核苷酸交换解偶联的突变,因此它们将 GPCR 捕获在活性状态构象中,相当于在激动剂结合的 GPCR 结构中观察到的与不含核苷酸的异源三聚体复合。Mini-G 蛋白可用于 G 蛋白 α 亚基的四个家族中的每一个。已经报道了用于评估 mini-G 与 GPCR 相互作用的生化分析(Nehmé等人,2017 年),包括使用固定受体的荧光饱和结合分析,以及基于荧光检测大小排阻色谱 (FSEC) 技术的分析。 Kawate 和 Gouaux,2006 年)。这些检测适用于洗涤剂溶解的裂解物中未纯化的受体,并使用 GFP 标记的 mini-G 进行检测。
我们最近报道了一种基于高分辨率透明天然电泳 (hrCNE) 技术的 GPCR-mini-G 偶联的新型生化分析,这是一种与荧光标记蛋白兼容的膜蛋白天然 PAGE 方法(Wittig等,2007) . 我们在工作过程中开发了该检测方法,以了解降钙素基因相关肽和肾上腺髓质素肽激动剂和 RAMP 辅助蛋白如何控制 B 类 GPCR 降钙素受体样受体 (CLR) 的 G 蛋白偶联(Roehrkasse等人,2020 年) )。该方法既可以快速、经济地筛选洗涤剂稳定的 GPCR-G 蛋白复合物,也可以对激动剂依赖性受体-mini-G 偶联进行定量研究。它不需要纯化的受体。相反,它使用在哺乳动物 HEK293S GnT1 –细胞系中瞬时过表达的 EGFP 标记受体。在筛选格式中,贴壁细胞培养物直接用洗涤剂溶解,经过简单的离心步骤,上清液通过非变性 PAGE 进行分析,并通过凝胶内荧光成像对受体进行可视化。外源性添加激动剂肽和纯化的 mini-G 会产生表明复合物形成的迁移率变化。在定量分析形式中,粗制膜制备用于提高重现性和增加通量。在第一种定量形式中,激动剂在存在恒定过量的 mini-G 的情况下发生变化,以生成结合曲线,该曲线测量激动剂对 mini-G 偶联受体的表观亲和力。在第二种定量格式中,mini-G 在受体饱和浓度恒定的激动剂存在下发生变化,以产生结合曲线,该曲线测量 mini-G 对激动剂占据的受体的表观亲和力。第二种格式提供了激动剂功效的部分测量(Roehrkasse等,2020)。
天然 PAGE 方法被证明是一种相对简单、廉价且用途广泛的检测方法,这使我们能够研究激动剂和 G 蛋白与 CLR:RAMP 复合物相互作用的生物化学。此处详细描述了筛选和定量分析格式。我们还发现该方法在热稳定性格式中非常有用,这里没有描述,但可以在我们的原始报告中找到(Roehrkasse等,2020)。该测定应适用于其他 GPCR,并且可能对其他具有挑战性的膜蛋白及其结合伙伴的生化研究有价值。

关键字:Mini-G, 膜蛋白, G蛋白偶联受体, 活性电泳, 肽激动剂, 热稳定性, 去污剂溶解, hrCNE

材料和试剂

 

1.     塑料制品

15 ml锥形管(VWR,目录号:76176-950

48 孔细胞培养板(CorningCostar,目录号:3548

离心管(VWR,目录号:87003-294

T-75培养瓶(Corning,目录号:43064

2.     细胞系

HEK293S GnT1 – ATCC,目录号:CRL-3022)(见注3

3.     细胞培养试剂

含有 4.5 g/L 葡萄糖和 L-谷氨酰胺的 DMEMLonza,目录号:12-604Q

胎牛血清(FBS)(Gibco,目录号:16000-044

非必需氨基酸(NEAA)(Gibco,目录号:11140-050

青霉素/链霉素(Gibco,目录号:15140-122

磷酸盐缓冲盐水(PBS)(Gibco,目录号:10010-023

聚乙烯亚胺,支化(PEI)(Sigma-Aldrich,目录号:408727-100 ml

4.     - 储存在室温下

过硫酸铵(APS)(Bio-Rad,目录号:1610700

氯化钙(CaCl )(Sigma-Aldrich,目录号:223506-500G

HEPES钠盐(Sigma-Aldrich,目录号:H7006-500G

氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G

氯化镁(MgCl )(Sigma-Aldrich,目录号:442611-500G

氯化钠(NaCl)(EMD Millipore,目录号:SX0420-5

丙戊酸钠盐(Sigma-Aldrich,目录号:P4543-25G

5.     醇类

异丙醇(VWRBDH,目录号:BDH1133-4LG

甲醇(SupelcoEMD Millipore,目录号:MX0475-1

6.     增溶化学品 - 储存于 -20°C

月桂基麦芽糖新戊二醇(LMNG)(Anatrace,目录号:NG310-25GM

胆固醇半琥珀酸酯(CHS)(Anatrace,目录号:CH210-5GM

7.     其他化学品

6-氨基己酸(Sigma-Aldrich,目录号:07260-1KG

30% 丙烯酰胺/双丙烯酰胺 29:1Acryl/Bis)(Bio-Rad,目录号:1610156

无脂肪酸 BSAFAF BSA)(PAA Laboratories,目录号:K35-002

氧化谷胱甘肽(GSSG)(Sigma-Aldrich,目录号:G4376-10G

谷胱甘肽还原(GSH)(Sigma-Aldrich,目录号:G4251-25G

咪唑(EMD Millipore,目录号:5720-500GM

氢氧化钠(NaOH)(VWRBDH,目录号:BDH3247-1

四亚甲基二胺(TEMED)(Bio-Rad,目录号:161-0800

TricineSigma-Aldrich,目录号:T0377-250G

蛋白酶抑制剂片剂(Thermo ScientificPierce,目录号:A32955

8.     肽类

α降钙素基因相关肽(Bachem,目录号:4013281

人肾上腺髓质素(Bachem,目录号:4034489

人肾上腺髓质素2/中间体(Bachem,目录号:4044529

9.     酵素

ApyraseNew England BioLabs,目录号:M0398L

10.  膜制剂和纯化的蛋白质

HEK293S GnT1 的粗膜制备——使用 pHLsec 表达载体瞬时共表达标记的 MBP-CLR-EGFP MBP-RAMP 的细胞(Aricescu2006);制备方法发表于 Roehrkasse等人(2020) – 在液氮中速冻并储存在 -80°C。最终制剂储存在 25 mM HEPESpH 7.510% (v/v) 甘油、25 mM NaCl2 mM MgCl 2 1x 蛋白酶抑制剂片剂中。蛋白质浓度为 ~4-6 mg/ml,受体为 ~100 nM

纯化的-SUMO-mini-G 蛋白(使用了 mini-G mini-G s/i mini-G s/q ;本协议利用来自 miniG示例数据);Carpenter Tate2016 年和 2017 年)、Nehmé等人发表的方法(2017) Roehrkasse等人(2020) – 储存在 -80°C。纯化的蛋白质以 ~25-30 mg/ml 储存在 25 mM HEPESpH 7.550% (v/v) 甘油、150 mM NaCl0.5 mM DTT1 mM MgCl 2 1 μM GDP的缓冲液中(见注释 1 2)。

11.  高分辨率透明天然 PAGE 凝胶(参见配方)

12.  ×结合缓冲液(见配方)

13.  洗涤剂缓冲液(见配方)

14.  运行缓冲区(见配方)

15.  培养基(见食谱)

16.  转染培养基(见配方)

17.  100 mM GSH GSSG 氧化还原缓冲液(见配方)

 

设备

 

1.     Mini-PROTEAN 电泳仪(Bio-Rad,型号:1658001FC

2.     Chemidoc MP 成像仪(Bio-Rad,型号:12003154

3.     CO 2培养箱(NuAire,型号:NU-5800

4.     成像托盘(Bio-Rad,目录号:12003028

5.     摇摆器(Reliable Scientific,型号:55

6.     管不倒翁(Labnet International,型号:H5500

7.     台式微型离心机(Eppendorf,型号:5415R

 

软件

 

1.     ImageLabBio-Radhttps: //tinyurl.com/2v4t44re 

2.     棱镜(GraphPadhttps ://www.graphpad.com/ 

 

程序

 

A.     制备:高分辨率透明天然 PAGE (hrCNE) 凝胶(Wittig2007)和缓冲液

1.     根据下面的配方部分准备 hrCNE 凝胶(参见配方 1,表 2)。我们使用了 8% 的分离胶,但也可以使用其他比例的分离胶(见注释 12)。

2.     根据以下配方(配方 2 3)准备×结合缓冲液和×增溶缓冲液库存。

3.     准备 hrCNE 20 ×阴极和 50 ×阳极缓冲液,并将它们在室温下储存在暗柜中(配方 4)。

 

B.     贴壁细胞培养——对于贴壁细胞培养筛选格式(见注 4

1.     制备:细胞培养基

a.     培养基(见配方 5)。

b.     转染培养基(参见配方 6)。

2.     播种

a.     种子 120,000 个细胞/ HEK293S GnT1——在经过细胞培养处理的48 孔透明板中加入 250 μl 培养基。

b.     将板在 37°C 下用 5% CO 2孵育24 小时,并生长至 ~90% 汇合度。

3.     使用 PEI 瞬时转染 DNA

制备质粒 DNA 转染混合物并添加到细胞中。

a.     原料

1)。DMEM(剩余体积共 30 μl)。

2)。受体表达质粒 DNA(每孔共 300 ng)(见注 5)。

3)。PEI DNA 的比例为 1.5:10.45 μl 1 mg/ml 原液)。

4)。46.67 mM 丙戊酸(2.8 μl 500 mM 储备液)。

b.     组装和添加到细胞

                                                        i.         对于转染混合物,将编号为 1)-3) 的成分组合到微量离心管中(添加顺序为 DMEM à DNA à PEI)并通过上下移液混合均匀。

                                                        ii.         在室温下孵育 10 分钟。

                                                        iii.         当转染混合物孵育时,小心地从 48 孔板中吸出培养基并更换为 250 μl 的转染培养基。

                                                        iv.         向离心管中加入丙戊酸(成分编号)并充分混合。

                                                        v.         30 μl 转染混合物转移到 48 孔板中的相应孔中,并通过轻轻旋转板混合。

                                                        vi.         30°C 下用 5% CO 2孵育板72 小时。

 

C.    准备:实验当天(筛选格式或定量格式)

1.     通过在 ddH O 中稀释 20× 50× 储备液来制备阴极和阳极缓冲液,并在运行天然凝胶之前在 4°C 下冷却几个小时,向阴极缓冲液中添加去污剂系统。

a.     阴极缓冲液(参见配方 4)。

b.     阳极缓冲液(参见配方 4)。

2.     在冰上解冻 4 × 结合缓冲液和 4 × 去污剂缓冲液、腺苷三磷酸双磷酸酶和肽激动剂。

3.     0.1 mg/ml FAF-BSA 制作结合缓冲液:对于 500 μl 总体积,将 372.5 μl ddH O 125 μl 4×结合缓冲液和 2.5 μl 20 mg/ml FAF-BSA(溶解在 ddH O 中)混合)。

4.     稀释 500 U/ml 腺苷三磷酸双磷酸酶原液

a.     筛选格式:在结合缓冲液中以 1:10 的比例加入 FAF-BSA 50 U/ml 并保持在冰上。

b.     定量格式:1:100 在结合缓冲液中加入 FAF-BSA 5 U/ml,并保持在冰上。

5.     使用 0.2 U/ml 腺苷三磷酸双磷酸酶制备洗涤剂缓冲液:对于 200 µl 总体积,将 199.2 μl 4× 洗涤剂缓冲液与 0.8 μl 50 U/ml 腺苷三磷酸双磷酸酶稀释液混合(见注释 6)。

6.     在设置检测之前,在冰上解冻膜制备和 mini-G 蛋白。

7.     制作100 mM GSH GSSG 氧化还原缓冲液(参见配方 7)(参见注释 7): 

8.     使用 100 mM GSH 100 mM GSSG 库存为特定检测格式制作工作库存。

a.     筛选形式

2.4 mM GSH/0.48 mM GSSG 库存(100 μl97.12 μl ddH O2.4 μl 100 mM GSH0.48 μl 100 mM GSSG)。

b.     定量格式

50 mM GSH/10 mM GSSG 原液(100 μl40 μl ddH O10 μl 100 mM GSSG50 μl 100 mM GSH)。

 

D.    贴壁细胞培养筛选格式

1.     结合反应             

最终反应由 25 μl 4× 去垢剂缓冲液和 75 μl 结合反应体积组成;在引入 48 孔板中的转染细胞之前,结合反应在微量离心管中组装。

a.     配料(按添加顺序)

1)。结合缓冲液(剩余体积共 75 μl)。

2)。200 μM GSH/40 μM GSSG6.25 μl 2.4 mM GSH/0.48 mM GSSG 储备液)。

3)。4% v/v 甘油(6 μl,来自 50% v/v 储备液)。

4)。66.66 μM mini-G 蛋白(体积取决于 mini-G 库存浓度)。

5)。13.33 μM 肽激动剂(体积取决于肽储备浓度)。

b.     组装和添加到细胞

                                                        i.         将编号为 1)-4) 的成分混合并搅拌均匀。

                                                        ii.         在冰上孵育 30 分钟,以减少 mini-G 蛋白。

                                                        iii.         将肽激动剂(成分 5)添加到所需的反应中并充分混合。

                                                        iv.         48 孔板中的细胞中吸取培养基。

                                                        v.         250 μl 1× PBS 清洗孔。

                                                        vi.         吸出 PBS 并将板置于冰上。

                                                       vii.         向每孔细胞中加入 75 μl 结合反应液,旋动板混匀。

                                                      viii.         48 孔板在冰上孵育 30 分钟。

2.     增溶

a.     向每个孔中加入 25 μl 4× Detergent 缓冲液 ± 0.2 U/ml 腺苷三磷酸双磷酸酶,并旋转板混合(见注释 6)。

b.     将板放在 4°C 的摇杆上,让细胞溶解 2 小时。

c.     移液器将裂解物移至冰上预冷的离心管中。

d.     使用 hrCNE 凝胶电泳和图像凝胶进行分析,如程序 FG 中所述。

 

E.     定量分析格式

1.     定量分析结合格式 1(图 1——激动剂对 mini-G 偶联受体的表观亲和力

a.     最终反应由 ¼ 4× 去污剂缓冲液和 ¾ 体积结合反应组成;结合反应由 mastermix 和激动剂系列稀释组合而成,以产生最终所需的浓度(每个步骤的浓度见表 1)。

b.     为了最大限度地提高重现性,将在所有反应中均等的反应组分组合在一个 mastermix (MM)(受体膜制备、过量的 mini-G 蛋白和腺苷三磷酸双磷酸酶)中;GSH GSSG mini-G 一起添加以保持 mini-G 蛋白减少(见注 7)。

c.     主混音:

                                       i.         成分(按添加顺序显示 400 μl MM 的体积)

1)。ddH O(剩余体积共 400 μl)。

2)。结合缓冲液(100 μl 4× 储备液)。

3)。0.1 mg/ml FAF-BSA2 μl 20 mg/ml)。

4)。400 μM GSH/80 μM GSSG3.2 μl 50 mM GSH/10 mM GSSG 储备液)。

5)。0.133 U/ml 腺苷三磷酸双磷酸酶(10.64 μl 5 U/ml 原液)(见注释 8)。

6). 133.5 μM mini-G 蛋白(体积取决于 mini-G 原液浓度)(见注 9)。

7)。26.67 nM 受体膜制剂(106.68 μl 100 nM 储备液)。

                                       ii.          部件

将编号为 1)-6) 的成分混合并搅拌均匀。

在冰上预孵育 30 分钟,以减少 mini-G 蛋白。

添加受体膜制剂并通过上下移液混合均匀。

d.     激动剂稀释:

                                                        i.         在含有 0.1 mg/ml FAF-BSA(浓度见表 1)的结合缓冲液中,在冰上的微量离心管中将激动剂肽稀释至最终所需最高浓度的 2.67 倍。

                                                        ii.         在含有 0.1 mg/ml FAF-BSA 结合缓冲液中连续稀释激动剂肽 3 倍(例如,在含有 0.1 mg/ml FAF-BSA 20 μl 1×结合缓冲液中稀释 10 μl 激动剂稀释液)。

e.     结合反应:通过将等体积 (15 μl + 15 μl) 的激动剂稀释液和 mastermix 结合来组装结合反应。

f.       在冰上孵育结合反应 30 分钟,然后进行增溶(步骤 E3)。

2.     定量分析结合格式 2 – mini-G 对激动剂占据的受体的表观亲和力

a.     最终反应由 1/4 4x 去污剂缓冲液和 3/4 结合反应组成,如测定格式 1;结合反应由 mastermix miniG 系列稀释组合而成,以产生最终所需的浓度(见表 1)。

b.     为了最大限度地提高重现性,将在所有反应中均等的反应组分组合在一个 mastermix (MM) [10 nM 的受体复合物,过量的 (10 μM) 激动剂(见注释 14]GSH GSSG 在连续稀释中与 mini-G 一起加入,以保持 mini-G 蛋白减少。

c.     主混音:

                                                                       i.         成分(按照显示的 400 μl MM 的体积添加顺序)

1)。ddH O(剩余体积共 400 μl)。

2)。结合缓冲液(100 μl 4× 储备液)。

3)。0.1 mg/ml FAF-BSA2 μl 20 mg/ml)。

4)。0.133 U/ml 腺苷三磷酸双磷酸酶(10.64 μl 5 U/ml 原液)(见注释 8)。

5)。26.67 μM 激动剂(体积取决于激动剂库存浓度)。

6). 26.67 nM 受体复合物(106.68 μl 100 nM 储备液)。

                                       ii.          按照指示的顺序组装所有成分并混合均匀。

d.     Mini-G 稀释液:

                                                        i.         在含有 0.1 mg/ml FAF-BSA 结合缓冲液中,将 mini-G 蛋白稀释至最终所需最高浓度 (200 μM) 2.67 倍。

                                                        ii.         添加 3 倍摩尔过量的 GSH/GSSG (600 μM),混合均匀,并在冰上预孵育 30 分钟,以减少 mini-G

                                                        iii.         在含有 0.1 mg/ml FAF-BSA 结合缓冲液中连续稀释 mini-G 3 倍(例如,在含有 0.1 mg/ml FAF-BSA 20 μl 1×结合缓冲液中稀释 10 μl 激动剂稀释液)请注意,GSH/GSSG mini-G 蛋白按比例稀释。

e.     结合反应:通过结合等体积 (15 μl + 15 μl) mini-G 稀释液和 mastermix 来组装结合反应。

f.       在冰上孵育结合反应 30 分钟,然后进行增溶(步骤 E3)。

 

1. 定量分析格式概述。

描述了与程序 CG 和协议中的数据分析相对应的定量 hrCNE 凝胶测定格式的关键步骤。


1. 协议各个阶段的缓冲液和结合反应成分浓度。

1结合缓冲液:25 mM HEPESpH 7.5140 mM NaCl10 mM KCl1 mM MgCl 2 mM CaCl + Pierce 蛋白酶抑制剂 (PI)

GSH:GSSG 的摩尔比始终为 5:1,而对于 mini-G s mini-G s/q 而言GSH:miniG 的比例始终为 3:1 ;有关 mini-G s/i 其他信息,请参阅 Roehrkasse等人(2020)

 

3.     增溶

a.     通过上下移液混合解冻的 4x 洗涤剂缓冲液。

b.     在每个结合反应中加入 10 μl 4×洗涤剂缓冲液(配方 3);这将使所有浓度达到最终浓度的 1

c.     将微量离心管放在冷藏室 (4°C) 中的离心管上,让反应溶解 2 小时(见注 10)。

d.     最终缓冲条件:

25 mM HEPESpH 7.5                                                       

140 毫米氯化钠

10 毫米氯化钾                                         

1 毫米氯化镁2

2 mM CaCl 2

0.5% w/v LMNG/0.05% w/v CHS

Pierce 蛋白酶抑制剂,0.538×

注意:这仅来自缓冲液,最终溶液还包括来自 mini-G 储存缓冲液的成分,添加量因测定形式以及膜制备储存缓冲液而异。我们样品中甘油的最终浓度从在未添加 mini-G 蛋白的样品中为 1% v/v,在含有最高量 mini-G 蛋白的样品中为 3%

 

F.     凝胶电泳(见注释 11 12

1.     在冷藏室 (4°C) 中设置 hrCNE 凝胶,并使用程序 C 中制备的预冷阴极和阳极缓冲液。

2.     100 V 下预运行凝胶 20 分钟,让洗涤剂进入凝胶。

3.     将步骤 D2 E3 中的1.5 ml 离心管放入预冷至 4°C 的离心机中。

4.     微量离心机 (Eppendorf 5415R) 4°C 下以 16,100 × g离心10 分钟

5.     20 μl 的每个反应上清液装入孔中。这可能是一个具有挑战性的步骤,可能需要一些练习,因为没有使用加载染料。

6.     200 V 下运行凝胶 3.5 小时。

 

G.    通过凝胶内荧光成像凝胶

1.     轻轻地将凝胶与玻璃板分离,并在 ddH O 中冲洗 3 次。

2.     使用 BioRad ChemiDoc MP 成像仪成像,使用印迹/紫外线/无染色成像板。

3.     预设程序:用于 EGFP 荧光的 ProQ Emerald 488

4.     曝光时间因条带强度而异。对于定量凝胶,确定最佳曝光时间并同时对所有凝胶成像;80-120 秒是一个合理的起点。

 

H.    使用 Bio-Rad Image 实验室软件量化条带强度

1.     YouTube 上的 Bio-Rad 提供了教程视频:https : //www.youtube.com/watch?v= IV_P47ScoYo & list = PLrAEgIY86I6zytNeMPGBhvXnKIm5MS-6J & index =3& t =8s

2.     使用图像工具根据需要拉直/旋转图像。

3.     使用泳道和波段工具定义泳道并选择波段

a.     手动分配 10 个泳道并调整泳道以与凝胶对齐。

b.     使用添加波段功能为所有二聚体和四元复合波段选择波段。

c.     在条带不易看到的区域,您可以查看车道剖面工具以查看微弱的条带。

4.     使用泳道剖面图选择/调整谱带峰的宽度。

a.     通过使用背景选择,可以根据需要调整和排除背景。

b.     查看每个波段的宽度并根据需要进行调整。注意带宽,以确保它们是均匀的。

5.     使用分析数据功能导出数据。

 

数据分析

 

在对数刻度上绘制由密度测定法确定的调整后的体积对激动剂(格式 1)或迷你 G(格式 2)浓度。使用 3 4 参数逻辑方程通过非线性回归拟合激动剂和 mini-G 结合数据,并确定 pEC 50 (四元复合物外观)或 pIC 50值(二聚体消失),以确定表观结合亲和力(见注13)。可以绘制四元复合带的出现和二聚体带的消失。我们已经看到 pEC 50 pIC 50值之间的良好一致性,如果 mini-G/激动剂亲和力足够高以完全可视化从二聚体到四元复合物的转变。

 

笔记

 

1.     我们将 mini-G 蛋白作为 SUMO 融合蛋白生产,最初用于纯化目的,但我们发现对于某些受体复合物,融合蛋白比游离的 mini-G 蛋白效果更好,大概是因为它们的较大尺寸产生了更好的迁移率变化。可能需要测试不同形式的 mini-G 以找到最佳的移动性转变。

2.     已经报道了 mini-G 12 纯化(Nehmé2017)。我们没有在我们的分析中使用这种蛋白质,但我们假设它也能起作用。

3.     HEK293S GnT1 细胞系缺乏 N-乙酰氨基葡萄糖转移酶 I 活性,因此会产生均质的 N-聚糖,从而在凝胶中形成清晰的条带。

4.     在细胞培养罩中使用适当的无菌技术。

5.     我们使用在 N 端用麦芽糖结合蛋白 (MBP) 和在 C 端用 EGFP 标记的 CLRRAMP 亚基也在 N 端用 MBP 标记。MBP 似乎允许凝胶上有更明确的条带,但这在所有情况下可能不是必需的。此外,柔性受体 C 尾被截断。用于表达的载体是先前描述的 pHLsec 载体(Aricescu2006)。

6.     在含有 mini-G 的孔中使用 4x Detergent buffer + 0.2 U/ml apyrase。在所有其他孔中使用 4x Detergent buffer w/o apyrase

7.     mini-G 蛋白有几个暴露于溶剂的半胱氨酸残基,当以高浓度储存时,这些残基有形成分子间二硫键的趋势。我们使用 GSH/GSSG 氧化还原缓冲液来减少 mini-G,而不会损坏我们的受体和肽激动剂,它们包含几个二硫键。在某些情况下,可以更简单地使用低浓度的 DTT TCEP 来降低 mini-G

8.     在原始论文中,我们按照定量格式中的描述添加了腺苷三磷酸双磷酸酶,但此后我们已转向在增溶步骤中添加腺苷三磷酸双磷酸酶,如粘附筛选格式所述。

9.     我们使用了 25 50 μM 的最终 mini-G 浓度。

10.  对于我们研究的大多数相互作用,30 分钟的结合反应和 2 小时的溶解足以达到平衡,但在某些情况下可能需要更长的时间。这应该根据具体情况进行测试。

11.  为天然凝胶配备指定设备是有益的,以避免从以前的 SDS-PAGE 凝胶运行中可能残留的 SDS 残留。

12.  应优化用于 hrCNE 分离胶的丙烯酰胺百分比。对于我们的目的,8% 的分离凝胶效果很好。梯度凝胶也可以考虑。

13.  激动剂、GPCR G 蛋白之间的相互作用很复杂,并且在两个方向上都有变构效应。此外,在电泳过程中可能会扰乱平衡。由于这些原因,我们将来自定量格式的亲和力描述为表观结合亲和力

14.  应选择激动剂浓度以使受体饱和(如果可能,为100×K )。

 

食谱

 

1.     高分辨率透明天然 PAGE 凝胶(见表 2

a.     0.1 M 咪唑 (pH 7.0)/2 M 6-氨基己酸的储备溶液可以制备成更大的体积 (250 ml),并在室温下避光保存。

b.     解析凝胶百分比的选择将取决于要可视化的蛋白质复合物的大小。

c.     在室温下,在15 ml 锥形管中,将 ddH O0.1 M 咪唑(pH 7.0/2 M 6-氨基己酸和 30% Acryl:Bis 混合并混合均匀。

d.     加入 APS TEMED,充分混合并立即浇注分离胶。

e.     用异丙醇顶掉凝胶,让凝胶凝固。

f.       倒掉异丙醇并用纸巾轻轻擦拭干净。

g.     用分离胶组装浓缩胶,用 10 孔梳子浇铸,让凝胶凝固。

h.     凝胶可以用保鲜膜包裹的湿纸巾包裹在 4°C 下储存数天。


2.hrCNE 凝胶配方。

 

2.     结合缓冲液

100 mM HEPESpH 7.5560 mM NaCl40 mM KCl4 mM MgCl 8 mM CaCl + 1× Pierce 蛋白酶抑制剂 (PI) – 10 ml 库存

 

库存解决方案

1 M HEPES (pH 7.5) 库存

5 M 氯化钠

1 M氯化钾

1 M 氯化镁2

1 M 氯化钙2

HEPES 储存在 4°C

所有其他的都可以在室温下储存。

 

对于 10 ml 储备溶液

1 毫升 1 M HEPESpH 7.5

1.12 毫升 5 M 氯化钠

0.4 毫升 1 M 氯化钾

0.04 毫升 1 M 氯化镁2

0.08 毫升 1 M CaCl 2

7.36 毫升ddH O

1 Pierce TM Protease Inhibitor Mini Tablets1x in 10 ml

 

将所有成分与 Pierce 蛋白酶抑制剂片剂一起加入 15 ml 锥形管中并涡旋混合(蛋白酶抑制剂片剂难以溶解)。

1 ml 等分试样在 -20°C 下储存。

 

3.     洗涤剂缓冲液

25 mM HEPES, pH 7.5, 140 mM NaCl, 10 mM KCl, 1 mM MgCl , 2 mM CaCl , 2% w/v LMNG/0.2% w/v CHS

库存解决方案

与配方 2 中相同的储备溶液;洗涤剂储备:10% w/v LMNG/1% w/v CHS ddH O 中,在 -20°C 下以 1 ml 等分试样储存。

 

对于 10 ml 储备溶液

0.25 毫升 1 M HEPESpH 7.5

0.28 毫升 5 M 氯化钠

0.1 毫升 1 M KCl

0.01 毫升 1 M 氯化镁2

0.02 毫升 1 M CaCl 2

2 ml 10% w/v LMNG/1% w/v CHS

7.34 毫升ddH O

1 Pierce TM Protease Inhibitor Mini Tablets1x in 10 ml

 

除洗涤剂外的所有成分加入15 ml 锥形管中,加入 Pierce 蛋白酶抑制剂片剂并涡旋混合(蛋白酶抑制剂片剂难以溶解)。

加入洗涤剂,轻轻但彻底地混合混合。

1 ml 等分试样在 -20°C 下储存。

 

4.     运行缓冲区

阴极缓冲液:50 mM Tricine7.5 mM 咪唑,pH 7.00.01% w/v LMNG/0.001% w/v CHS

阳极缓冲液:25 mM 咪唑,pH 7.0

20× 阴极缓冲液储备:1 M Tricine150 mM 咪唑,pH 7.0

50× 阳极缓冲液:1.25 M 咪唑,pH 7.0

5.     文化传媒

DMEM

10% 胎牛血清

国家航空航天局

6.     转染培养基

DMEM

2% 胎牛血清

国家航空航天局

50 U/ml 青霉素

50 微克/毫升链霉素

7.     100 mM GSH GSSG 氧化还原缓冲液(见注 7

a.     称量 GSH GSSG 粉末并添加到单独的 15 ml 锥形管中。

b.     ddH O添加到最终所需浓度并涡旋溶解。

c.     通过添加 NaOH pH 值调整为中性。

致谢

 

这项工作得到了赠款 NIH R01GM104251 和长老会健康基金会种子赠款 (AAP) 以及 NIH 博士前 MD/PhD 奖学金 1F30 HL142232 (AMR) 的支持。这种方法最初是在 Roehrkasse人中描述的。(2020) 

 

利益争夺

 

没有作者有任何竞争利益要披露。

 

伦理

 

该协议使用标准的永生化人类细胞系,不涉及人类或动物受试者。

 

参考

1.     Aricescu, AR, Lu, W. Jones, EY (2006)在哺乳动物细胞中高水平蛋白质生产的时间和成本效益系统。Acta Crystallogr D Biol Crystallogr 62(第 10 篇):1243-1250

2.     Carpenter, B. Tate, CG (2016)设计最小 G 蛋白以促进 G 蛋白偶联受体在其活性构象中的结晶。Protein Eng Des Sel 29(12): 583-594

3.     Carpenter, B. Tate, CG (2017)大肠杆菌中表达和纯化 MiniG 蛋白。生物协议7(8)e2235

4.     Gregorio, GG, Masureel, M., Hilger, D., Terry, DS, Juette, M., Zhao, H., Zhou, Z., Perez-Aguilar, JM, Hauge, M., Mathiasen, S., et阿尔。(2017)β2AR-G 蛋白激活中配体功效的单分子分析。自然547(7661)68-73

5.     Huang, SK, Pandey, A., Tran, DP, Villanueva, NL, Kitao, A., Sunahara, RK, Sljoka, A. Prosser, RS (2021)描绘 G 蛋白偶联过程中腺苷 A2A 受体的构象景观。 细胞184(7)1884-1894 e1814

6.     Kawate, T. Gouaux, E. (2006)用于内膜蛋白预结晶筛选的荧光检测大小排阻色谱。结构14(4)673-681

7.     Nehmé, R., Carpenter, B., Singhal, A., Strege, A., Edwards, PC, White, CF, Du, H., Grisshammer, R. Tate, CG (2017)Mini-G 蛋白:研究 GPCR 活性构象的新工具。PloS 12(4): e0175642

8.     Pierce, KL, Premont, RT Lefkowitz, RJ (2002)七跨膜受体。Nat Rev Mol Cell Biol 3(9): 639-650

9.     Roehrkasse, AMWarnerML BooeJM Pioszak, AA2020 年)。使用天然 PAGE 测定法对 G 蛋白与降钙素基因相关肽和肾上腺髓质素受体偶联的生化表征。J Biol Chem 295(28): 9736-9751

10.  Sriram, K. Insel, PA (2018)G 蛋白偶联受体作为批准药物的靶点:有多少靶点和多少药物?Mol Pharmacol 93(4): 251-258

11.  Wittig, I.Karas, M. Schagger, H. (2007)高分辨率清晰的天然电泳,用于膜蛋白复合物的凝胶功能测定和荧光研究。Mol 细胞蛋白质组学6(7)1215-1225

12.  Yao, XJ, Velez Ruiz, G., Whorton, MR, Rasmussen, SG, DeVree, BT, Deupi, X., Sunahara, RK Kobilka, B. (2009)配体功效对 GPCR-G 蛋白复合物形成和稳定性的影响。Proc Natl Acad Sci USA 106(23): 9501-9506

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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Roehrkasse, A. M., Karim, J. A. and Pioszak, A. A. (2021). A Native PAGE Assay for the Biochemical Characterization of G Protein Coupling to GPCRs. Bio-protocol 11(24): e4266. DOI: 10.21769/BioProtoc.4266.
  2. Roehrkasse, A. M., Warner, M. L. Booe, J. M., and Pioszak, A. A. (2020). Biochemical characterization of G protein coupling to calcitonin gene-related peptide and adrenomedullin receptors using a native PAGE assay. J Biol Chem 295(28): 9736-9751.
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