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Sep 2021
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In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions
评估蛋白质-磷脂相互作用的特异性和亲和力的体外测定   

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Abstract

Protein–lipid interactions play important roles in many biological processes, including metabolism, signaling, and transport; however, computational and structural analyses often fail to predict such interactions, and determining which lipids participate in these interactions remains challenging. In vitro assays to assess the physical interaction between a protein of interest and a panel of phospholipids provide crucial information for predicting the functionality of these interactions in vivo. In this protocol, which we developed in the context of evaluating protein–lipid binding of the Arabidopsis thaliana florigen FLOWERING LOCUS T, we describe four independent in vitro experiments to determine the interaction of a protein with phospholipids: lipid–protein overlay assays, liposome binding assays, biotin-phospholipid pull-down assays, and fluorescence polarization assays. These complementary assays allow the researcher to test whether the protein of interest interacts with lipids in the test panel, identify the relevant lipids, and assess the strength of the interaction.

Keywords: Protein-phospholipids interaction (蛋白质-磷脂相互作用), In vitro assay (体外试验), FLOWERING LOCUS T (FLOWERING LOCUS T), Phosphatidylglycerol (磷脂酰甘油), Phosphatidylcholine (磷脂酰胆碱), Phospholipids (磷脂), Liposome (脂质体), Fluorescence polarization (光偏振)

Background

Protein–lipid interactions form the basis of many cellular functions. In particular, interactions involving lipids in the plasma membrane and other cellular membranes have essential functions in transport, signaling, and metabolism. Proteins also interact with lipids in lipid biosynthesis; moreover, lipid catabolism provides energy for animals and for seed germination in many plants. Despite the importance of protein–lipid interactions, many such interactions are not predicted from protein sequences and structural analysis (Yao et al., 2013; Kim et al., 2019). Moreover, identifying the specific lipid(s) involved in these interactions remains challenging.


In addition to their well-known roles in other processes, protein–phospholipid interactions modulate flowering time in Arabidopsis thaliana (Nakamura et al., 2014; Susila et al., 2021). In this process, FLOWERING LOCUS T (FT) protein specifically interacts with phosphatidylglycerol (PG) and is sequestered into the membrane to prevent precocious flowering, especially at low ambient temperature (Susila et al., 2021). FT belongs to the phosphatidylethanolamine binding protein (PEBP) family and has a conserved anion-binding pocket that could be responsible for binding PG (Bernier et al., 1986; Jin et al., 2021). Lipid binding also affects other regulatory factors; for example, several transcription factors without transmembrane domains interact with phosphatidic acid (PA) to regulate plant development and stress responses (Yao et al., 2013; Kim et al., 2019). Research on phospholipid-binding proteins may uncover additional novel regulatory mechanisms in complex biological systems.


Here, we describe four methods that we used to screen for interactions of FT with phospholipids: 1) lipid–protein overlay assays, 2) liposome binding assays, 3) biotin-phospholipid pull-down assays, and 4) fluorescence polarization assays (Susila et al., 2021). We believe that using different in vitro methods is necessary to validate the protein–phospholipid interactions. This versatile protocol can be used for any protein of interest, although the incubation temperature and the duration of incubation should be tested for each protein. These assays provide complementary information on protein–lipid interactions, including whether the protein of interest interacts with the lipids in the test panel, which lipid(s) it prefers, and how strongly it interacts with each species of lipid.

Materials and Reagents

  1. Materials

    Recombinant FT protein was purified from Escherichia coli following standard methods (Susila et al., 2021). The phospholipids and galactolipids were obtained from Sigma-Aldrich or Avanti Polar Lipids (Table 1).


    Table 1. Lipids used in this study

    Lipid Abbreviation Vendor Catalog number
    1,2-dipalmitoyl-sn-glycero-3-phosphate DPPA Avanti Polar Lipids 830855P
    1,2-dipalmitoyl-sn-glycero-3-phospho-1'-myo-inositol) DPPI Avanti Polar Lipids 850141P
    1,2-dipalmitoyl-sn-glycero-3-phospho-1'-rac-glycerol) DPPG Avanti Polar Lipids 840455P
    1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine DPPS Avanti Polar Lipids 840037P
    1,2-dipalmitoyl-sn-glycero-3-phosphocholine DPPC Avanti Polar Lipids 850355
    1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine DPPE Avanti Polar Lipids 850705
    L-α-phosphatidyl-DL-glycerol ammonium salt from egg yolk lecithin PG Sigma-Aldrich P0514
    L-α-phosphatidylethanolamine from egg yolk PE Sigma-Aldrich P7943
    1-oleoyl-2-[12-biotinyl(aminododecanoyl)]-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) tBiotin-PG Avanti Polar Lipids 860581C
    1-oleoyl-2-[12-biotinyl(aminododecanoyl)]-sn-glycero-3-phosphocholine tBiotin-PC Avanti Polar Lipids 860563C


  2. Lipid–protein overlay assay

    1. Volac Pasteur pipette (Sigma-Aldrich, catalog number: Z310727)

    2. Polyvinylidene fluoride (PVDF) membrane (Millipore, catalog number: IPVH304F0)

    3. X-ray film (AGFA, catalog number: CP-BU New)

    4. Chloroform (Merck, catalog number: 1.02445.1000)

    5. Fatty acid-free bovine serum albumin (Sigma-Aldrich, catalog number: A6003)

    6. AntiHis monoclonal antibody (Sigma-Aldrich, catalog number: H1029; 1:2,000 dilution)

    7. Anti-Strep-tag II monoclonal antibody (Abcam, catalog number: ab76949; 1:2,000 dilution)

    8. Goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: A28177; 1:10,000 dilution)

    9. Western Blotting Detection Reagent Kit Ab Signal (AbClon, catalog number: Abc-3001)

    10. Tris (Duchefa, catalog number: T1501)

    11. NaCl (Duchefa, catalog number: S0520)

    12. Glycerol (Duchefa, catalog number: G1345)

    13. 2-mercaptoethanol (Sigma-Aldrich, catalog number: M6250)

    14. Purified tagged protein (His-FT or FT-StII in dialysis buffer; see Recipes)

    15. Tris-buffered saline (TBS) (see Recipes)


  3. Liposome binding assay

    1. Volac Pasteur pipette (Sigma-Aldrich, catalog number: Z310727)

    2. Protein LoBind Tubes (Eppendorf, catalog number: 0030108116)

    3. PVDF membrane (Millipore, catalog number: IPVH304F0)

    4. X-ray film (AGFA, catalog number: CP-BU New)

    5. Chloroform (Merck, catalog number: 1.02445.1000)

    6. Skim milk (Sigma-Aldrich, catalog number: 70166)

    7. Bovine serum albumin (MP Biomedicals, catalog number: 0216006980)

    8. AntiHis monoclonal antibody (Sigma-Aldrich, catalog number: H1029; 1:2,000 dilution)

    9. Goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: A28177; 1:10,000 dilution)

    10. Western blotting Detection Reagent Kit Ab Signal (AbClon, catalog number: Abc-3001)

    11. SDS (Sigma-Aldrich, catalog number: L5750)

    12. Bromophenol blue (Sigma-Aldrich, catalog number: B0126)

    13. Purified tagged protein (His-FT or FT-StII in dialysis buffer; see Recipes)

    14. Tris-buffered saline (TBS) (see Recipes)

    15. SDS sample buffer (see Recipes)


  4. Biotin-phospholipid pull-down assay

    1. Volac Pasteur pipette (Sigma-Aldrich, catalog number: Z310727)

    2. Protein LoBind Tubes (Eppendorf, catalog number: 0030108116)

    3. PVDF membrane (Millipore, catalog number: IPVH304F0)

    4. X-ray film (AGFA, catalog number: CP-BU New)

    5. Chloroform (Merck, catalog number: 1.02445.1000)

    6. Ethanol (Merck, catalog number: 1.00983.1011)

    7. Dynabeads MyOne Streptavidin T1 (Invitrogen, catalog number: 65601)

    8. Skim milk (Sigma-Aldrich, catalog number: 70166)

    9. Bovine serum albumin (MP Biomedicals, catalog number: 0216006980)

    10. AntiHis monoclonal antibody (Sigma-Aldrich, catalog number: H1029; 1:2,000 dilution)

    11. Goat anti-mouse IgG (Thermo Fisher Scientific, catalog number: A28177; 1:10,000 dilution)

    12. Western blotting Detection Reagent Kit Ab Signal (AbClon, catalog number: Abc-3001)

    13. SDS sample buffer (see Recipes)

    14. Purified tagged protein (His-FT in dialysis buffer; see Recipes)

    15. Tris-buffered saline (TBS) (see Recipes)


  5. Fluorescence polarization assay

    1. Volac Pasteur pipette (Sigma-Aldrich, catalog number: Z310727)

    2. Protein LoBind Tubes (Eppendorf, catalog number: 0030108116)

    3. 96-well black flat-bottom polystyrene plate (Corning, catalog number: 3915)

    4. PierceTM NHS-Rhodamine Antibody Labeling Kit (ThermoFisher Scientific, catalog number: 53031)

    5. Chloroform (Merck, catalog number: 1.02445.1000)

    6. KCl (Sigma Aldrich, catalog number: P3911)

    7. Na2HPO4 (Sigma-Aldrich, catalog number: S9763)

    8. KH2PO4 (Sigma-Aldrich, catalog number: P0662

    9. Purified tagged protein (His-FT in PBS dialysis buffer; see Recipes)

    10. Phosphate-buffered saline (PBS) (see Recipes)

    11. PBS elution buffer (see Recipes)

Equipment

  1. Lipid–protein overlay assay

    1. Amber glass vial (Shimadzu Scientific Korea, catalog number: 91005-0920)

    2. Vortex mixer (Scientific Industries, Genie 2, model: G-560)

    3. Micropipette (Eppendorf Research, catalog number: 3123000918)

    4. Western blot tray

    5. Orbital shaker (Best of Lab Equipments, model: RF300)

    6. X-ray film cassette

    7. X-ray film processor (Healthcare Solutions Inc., model: JP-33)


  2. Liposome binding assay

    1. Amber glass vial (Shimadzu Scientific Korea, catalog number: 91005-0920)

    2. Vortex mixer (Scientific Industries, Genie 2, model: G-560)

    3. Micropipette (Eppendorf Research, catalog number: 3123000918)

    4. Bioruptor sonication device (Bio-Medical Science, model: KRB 01)

    5. Refrigerated centrifuge (Eppendorf Research, model: 5430R)

    6. Incubator (Vision Scientific, model: VS-8480MX2-DT)

    7. Heat block (Best of Lab Equipments, model: CF1)

    8. Western blot tray

    9. Orbital shaker (Best of Lab Equipments, model: RF300)

    10. X-ray film cassette

    11. X-ray film processor (Healthcare Solutions Inc., model: JP-33)


  3. Biotin-phospholipid pull-down assay

    1. Amber glass vial (Shimadzu Scientific Korea, catalog number: 91005-0920)

    2. Vortex mixer (Scientific Industries, Genie 2, model: G-560)

    3. Micropipette (Eppendorf Research, catalog number: 3123000918)

    4. Tube rotator (Seoulin Bioscience, model: SLRM-3)

    5. Magnetic separation rack

    6. Spin-down centrifuge (Allsheng, model: mini-6K)

    7. Heat block (Best of Lab Equipments, model: CF1)

    8. Western blot tray

    9. Orbital shaker (Best of Lab Equipments, model: RF300)

    10. X-ray film cassette

    11. X-ray film processor (Healthcare Solutions Inc., model: JP-33)


  4. Fluorescence polarization assay

    1. Amber glass vial (Shimadzu Scientific Korea, catalog number: 91005-0920)

    2. Vortex mixer (Scientific Industries, Genie 2, model: G-560)

    3. Micropipette (Eppendorf Research, catalog number: 3123000918)

    4. Bioruptor Sonication device (Bio-Medical Science, model: KRB 01)

    5. Refrigerated centrifuge (Eppendorf Research, model: 5430R)

    6. Incubator (Vision Scientific, model: VS-8480MX2-DT)

    7. SpectraMax Multi-Mode Plate Reader (Molecular Devices)

Software

  1. ImageJ Fiji, an open source software (Schindelin et al., 2012)

  2. GraphPad Prism 5 (Graph Pad Software Inc.)

Procedure

  1. Lipid–protein overlay assay

    In this qualitative assay, lipids are immobilized on a membrane and incubated with tagged protein; any bound protein is then detected by antibodies to the epitope tag (Figure 1 and Figure 5A). We recommend the use of a tag protein (e.g., GST, GFP, or MBP) or a protein without lipid-binding capacity as a negative control for the experiment.



    Figure 1. Lipid–protein overlay assay.

    Lipids are immobilized on a PVDF membrane and incubated with the protein of interest. Lipid-bound proteins are detected with western blotting.


    1. Dissolve the lipids in pure chloroform to prepare stock solutions (1 mg/mL). Use a Volac Pasteur pipette to mix and transfer the lipid solutions.

    2. Store the lipid stock solutions in amber glass vials at -20°C.

    3. Cut the PVDF membrane (2 × 8 cm) and mark the spots (at least 1 cm apart) for the lipids.

    4. Slowly spot 5 µg of each lipid (5 µL of stock solution) onto the prepared PVDF membrane (at least 1–2-mm diameter) and dry the membrane for at least 30 min to 1 h at room temperature.

    5. Place the membrane in the western blot tray (6 × 10 cm) and incubate with 10 mL of blocking solution (TBS containing 3% [w/v] fatty acid-free bovine serum albumin), at room temperature for 1 h with gentle agitation (10 rpm).

    6. Discard the blocking solution and incubate the membrane with 60 µg of recombinant His-FT or FT-StII protein in 5 mL of blocking solution at 4°C for 16 h with gentle agitation (10 rpm).

    7. Discard the protein solution and wash the membrane with 10 mL of TBS for 5 min; repeat the washes a total of four times.

    8. Incubate the membrane with 10 mL blocking solution containing the primary antibody, either antiHis monoclonal antibody or Anti-Strep-tag II monoclonal antibody, at room temperature for 1 h with gentle agitation (10 rpm).

    9. Discard the antibody solution and wash the membrane with 10 mL of TBS for 5 min; repeat the washes four times.

    10. Incubate the membrane with 10 mL of blocking solution containing goat anti-mouse IgG secondary antibody at room temperature for 1 h with gentle agitation (10 rpm).

    11. Discard the antibody solution and wash the membrane with 10 mL of TBS solution for 5 min; repeat the washes four times.

    12. Incubate the membrane with 2 mL of horseradish peroxidase (HRP) substrate solution for 5 min.

    13. Remove the excess liquid from the membrane and put the membrane wrapped in plastic in an X-ray film cassette, and detect the signal using the X-ray film with an X-ray film processor.


  2. Liposome binding assay

    In this qualitative assay, tagged protein is incubated with liposomes composed of different ratios of two lipids; bound protein is pulled down with the liposomes and detected by western blotting (Figure 2 and Figure 5B). We recommend the reader to use a tag protein (e.g., GST, GFP, or MBP) or a protein without lipid-binding capacity as a negative control for the experiment.



    Figure 2. Liposome binding assay.

    A protein-of-interest is incubated with liposomes composed of different ratios of two lipids. Liposome-bound proteins are precipitated and detected by western blotting.


    1. Dissolve the lipids in pure chloroform to prepare stock solutions (1 mg/mL). Use a Volac Pasteur pipette to mix and transfer the lipid solution.

    2. Store the lipid stock solutions in amber glass vials at -20°C.

    3. To make the liposomes, transfer the phospholipid solutions into protein LoBind tubes in the desired ratios, with 50 µg lipids per reaction. For example, PG/PC liposome: 50/0 µg, 40/10 µg, 30/20 µg, 20/30 µg, 10/40 µg, and 0/50 µg.

    4. Evaporate the chloroform by leaving the tube open to obtain a lipid film in each tube.

    5. Rehydrate the lipid film with 100 µL of TBS and incubate at 37°C for 1 h.

    6. Vortex to disperse the lipids for 10 min.

    7. Sonicate the lipid mixture (high setting: 320W) for 10 min (30 s on and 30 s off) in an ice-water bath.

    8. Replace the ice-water from the water bath and repeat step B7.

    9. Centrifuge the liposomes for 10 min at 20,000 × g and 4°C.

    10. Discard the supernatant, and wash the liposomes with 100 µL of cold TBS twice.

    11. Resuspend the liposomes in 50 µL of cold TBS (final concentration of 1 mg/mL).

    12. Add 1 µg of purified protein (His-FT) to the liposome solution and incubate at 30°C for 30 min with gentle agitation. Use one tube without liposomes (only TBS) as a negative control.

    13. Centrifuge the mixture for 10 min at 20,000 × g and 4°C.

    14. Remove the supernatant and store in another tube for western blot control.

    15. Wash the liposome twice with 100 µL of cold TBS solution. Vortex the mixture until all the pellet is completely resuspended.

    16. Add 10 µL of 6× SDS sample buffer to the pellet and supernatant from step B13 and incubate the sample at 95°C for 5 min in a heat block. Store the sample at -20°C until further use.

    17. Prepare 12% standard polyacrylamide gel for SDS-PAGE (mini gel: 10 × 8 cm; 11 wells).

    18. Load the sample into the well, run the gel with 110 V for 2 h, and transfer the protein onto a PVDF membrane.

    19. Place the membrane in the western blot tray and incubate with 10 mL of blocking solution (TBS containing 3% [w/v] skim milk), at room temperature for 1 h with agitation (50 rpm).

    20. Discard the blocking solution and briefly wash the membrane with ddH2O.

    21. Incubate the membrane with the primary antibody, anti-His monoclonal antibody, in 10 mL of TBS containing 1% (w/v) bovine serum albumin at 4°C overnight.

    22. Discard the antibody solution and wash the membrane with 10 mL of TBS solution for 5 min four times.

    23. Incubate the membrane with the goat anti-mouse IgG secondary antibody in 10 mL of TBS containing 1% (w/v) bovine serum albumin at room temperature for 1 h.

    24. Discard the antibody solution and wash the membrane with 10 mL of TBS solution for 5 min four times.

    25. Incubate the membrane with 2 mL of HRP substrate solution for 5 min.

    26. Remove the excess liquid from the membrane and put the membrane wrapped in plastic in an X-ray film cassette, and detect the signal using the X-ray film with an X-ray film processor.


  3. Biotin-phospholipid pull-down assay

    In this qualitative assay, biotinylated lipids are incubated with protein; the lipids are pulled down with streptavidin beads, and bound protein is detected by western blotting (Figure 3 and Figure 5C).



    Figure 3. Biotin-phospholipid pull-down assay.

    Biotin-labeled phospholipids are incubated with a protein-of-interest. The protein-lipids complexes are pulled down by streptavidin beads. The lipid-bound proteins are detected by western blotting.


    1. Dissolve the biotin-labeled lipids in pure chloroform to prepare stock solutions (1 mg/mL). Use a Volac Pasteur pipette to mix and transfer the lipid solutions.

    2. Store the lipid stock solutions in amber glass vials at -20°C.

    3. Transfer 15 µL (15 µg) of each biotin-labeled phospholipid into a protein LoBind tube, and evaporate the chloroform to obtain a lipid film.

    4. Rehydrate the lipid film with 10 µL of 50% ethanol.

    5. Incubate the lipid solution at room temperature for 15 min with brief vortexing every 5 min.

    6. Add 40 µL of TBS containing 1 µg of purified protein to the lipid solution.

    7. Use 50 µL of TBS containing 1 µg of purified protein (without lipid solution) as a negative control.

    8. Incubate at 30°C for 30 min with gentle agitation.

    9. During the incubation in step C7, prepare the beads by transferring 20 µL of Dynabeads MyOne Streptavidin T1 to protein LoBind tubes.

    10. Collect the beads with the magnetic separation rack, and wash the beads twice with 500 µL of TBS containing 0.5% (w/v) bovine serum albumin.

    11. Resuspend the beads in 450 µL of TBS containing 0.5% (w/v) bovine serum albumin.

    12. Spin down the lipid–protein mixture from step C7 briefly, put 5 µL of the supernatant into a new tube as an input control (10% input), and transfer the rest of the mixture into the tube containing the beads. Store the input sample at -20°C.

    13. Incubate the bead–lipid–protein mixture in a tube rotator (10 rpm) at 4°C overnight.

    14. Collect the beads with the magnetic separation rack and wash the beads twice with 500 µL of TBS containing 0.5% (w/v) bovine serum albumin.

    15. Collect the beads from the last washing step and add the SDS sample buffer to the beads and the input samples from step C11. Incubate the samples at 95°C for 5 min in a heat block. Store the samples at -20°C.

    16. Prepare a standard 12% polyacrylamide gel for SDS-PAGE.

    17. Load the sample into the well, run the gel with 110 V for 2 h, and transfer the protein onto a PVDF membrane.

    18. Place the membrane in the western blot tray and incubate with 10 mL of blocking solution (TBS containing 3% [w/v] skim milk) at room temperature for 1 h with gentle agitation (50 rpm).

    19. Discard the blocking solution and briefly wash the membrane with ddH2O.

    20. Incubate the membrane with the primary antibody, antiHis monoclonal antibody, in 10 mL of TBS containing 1% (w/v) bovine serum albumin at 4°C overnight.

    21. Discard the antibody solution and wash the membrane with 10 mL of TBS for 5 min; repeat the washes four times.

    22. Incubate the membrane with the goat anti-mouse IgG secondary antibody in 10 mL of TBS containing 1% (w/v) bovine serum albumin at room temperature for 1 h.

    23. Discard the antibody solution and wash the membrane with 10 mL of TBS solution for 5 min; repeat the washes four times.

    24. Incubate the membrane with 2 mL of HRP substrate solution for 5 min.

    25. Remove the excess liquid from the membrane and put the membrane wrapped in plastic in an X-ray film cassette, and detect the signal using the X-ray film with an X-ray film processor.


  4. Fluorescence polarization assay

    In this quantitative assay, protein–lipid interactions alter the polarity of light emitted from a fluorophore attached to the protein of interest and excited with polarized light; graphing the resulting anisotropy values as a function of liposome concentration gives the protein–lipid binding affinity (Figure 4 and Figure 5D).



    Figure 4. Fluorescence polarization assay.

    Fluorescence dye-labeled protein are incubated with liposomes. The degree of the interaction is calculated through the value of fluorescence polarization or anisotropy.


    1. Dissolve the lipids in pure chloroform to prepare stock solutions (1 mg/mL). Use a Volac Pasteur pipette to mix and transfer the lipid solutions.

    2. Store the lipid stock solutions in amber glass vials at -20°C.

    3. Prepare 1 mg of purified protein (His-FT) in 500 µL of PBS dialysis buffer. Avoid any amine molecules such as Tris or glycine in the buffer.

    4. To label the protein, add the 500 µL of protein solution to a vial containing NHS-Rhodamine reagent from the kit and mix with pipette until all the dye dissolves.

    5. Incubate the vial at room temperature for 1 h in the dark.

    6. To remove the unbound dye, prepare two spin columns by resuspending the resin from the kit by vortexing and pipetting and then transfer 400 µL of resin to each spin column.

    7. Centrifuge the columns for 30 s at 1,000 × g at room temperature to remove the buffer.

    8. Put the column in the new collection tube and add 250 µL of protein–dye mixture from step D5 to the spin column.

    9. Mix the sample by vortexing and pipetting.

    10. Centrifuge the column for 30 s at 1,000 × g at room temperature.

    11. Collect and combine the flow-through from both collection tubes.

    12. Dilute the labeled protein mixture with PBS dialysis buffer to a concentration of 4 µM (equal to concentration of 0.1 µg/µL for His-FT) as a stock.

    13. Aliquot and store the labeled protein in a protein LoBind tube at -20°C in the dark until further use.

    14. To make the liposomes, transfer phospholipid solutions into microcentrifuge tubes to produce the desired concentration series, as described below. Calculate the amount of phospholipids needed prior to making stock solutions for the serial dilution analysis. Detailed information, including the molecular weight of phospholipids, can be obtained from the Avanti Polar Lipids website (https://avantilipids.com/).

      In this case, we used DPPG (MW: 744.492 g/mol) and DPPC (MW: 733.562 g/mol). We used serial dilutions of lipids with concentrations of 0, 10, 20, 30, 50, 75, 100, and 150 µM in 200-µL reaction volumes with three technical replicates (from 2 mM stock solution). Therefore, we transferred 298 µL of DPPG and 293 µL of DPPC stock solutions (1 mg/mL) to the microcentrifuge tube to make 200 µL of 2 mM stock liposome.

    15. Evaporate the chloroform to obtain a lipid film.

    16. Rehydrate the lipid film with 200 µL of PBS and incubate at 37°C for 1 h.

    17. Vortex the lipid dispersion for 10 min.

    18. Sonicate the samples (high setting) twice for 10 min (30 s on and 30 s off) in an ice-water bath.

    19. Centrifuge the liposome for 10 min at 20,000 × g and 4°C.

    20. Discard the supernatant and wash the liposomes with 200 µL of cold PBS twice.

    21. Resuspend the liposomes in 200 µL of cold PBS (final concentration of 2 mM).

    22. Prepare the 96-well plate and transfer 1.5 µL of protein, liposome, and PBS to each well. Set the total volume to 200 µL (Table 1). The liposome was diluted to concentrations of 0, 10, 20, 30, 50, 75, 100, and 150 µM, as mentioned above.


      Table 1. Composition of each component in the assay

      Component Liposome

      (0 µM)

      Liposome

      (10 µM)

      Liposome

      (20 µM)

      -----

      Liposome

      (150 µM)

      Protein (µL)

      Liposome (µL)

      PBS (µL)

      1.5 µL

      0 µL

      198.5 µL

      1.5 µL

      1 µL

      197.5 µL

      1.5 µL

      2 µL

      196.5 µL

      -----

      1.5 µL

      15 µL

      183.5 µL


    23. Incubate the plate at room temperature for 30 min with gentle agitation.

    24. Prepare the SpectraMax Multi-Mode Plate Reader. In the software interface, set temperature to 30°C, choose end point measurement with top read, choose Costar black 96-well with no lid as the assay plate, and choose auto mix for 3 s. Finally, set the excitation wavelength to 544 nm and the emission wavelength to 575 nm, with the cut-off set to 570 nm.

    25. Calculate the anisotropy (r) value using the software. The values can be saved to a text (.txt) file or recorded manually.

    26. Plot the obtained data (triplicates) into the scatter plot in the GraphPad Prism 5 software, with the liposome concentrations on the x-axis and the anisotropy (r) values on the y-axis.

    27. Analyze the data using the nonlinear regression model (one site, total binding) to obtain the dissociation constant to determine the binding affinity.

Data analysis

Representative data



Figure 5. Representative results of in vitro FT protein–phospholipid interaction assays.

A. Lipid–protein overlay assay. B. Liposome binding assay. C. Biotin-phospholipid pull-down assay. D. Fluorescence polarization assay. DPPI: dipalmitoylphosphatidylinositol, DPPA: dipalmitoylphosphatidic acid, DPPS: dipalmitoylphosphatidylserine, DPPG: dipalmitoylphosphatidylglycerol, DPPE: dipalmitoylphosphatidylethanolamine, DPPC: dipalmitoylphosphatidylcholine, PG: phosphatidylglycerol, PE: phosphatidylethanolamine, PC: phosphatidylcholine.

Recipes

The water used in the following recipes is double-distilled water (ddH2O).

  1. Dialysis buffer (pH 8.0)

    50 mM Tris

    150 mM NaCl

    5% glycerol

    Filter sterilize and add 5 mM 2-mercaptoethanol immediately prior to use.

  2. Tris-buffered saline (TBS) (pH 7.2)

    50 mM Tris

    150 mM NaCl

    Filter sterilize.

  3. 4× SDS sample buffer

    200 mM Tris-Cl pH 6.8

    8% SDS

    0.4% bromophenol blue

    40% glycerol

  4. Phosphate-buffered saline (PBS) (pH 7.4)

    137 mM NaCl

    2.7 mM KCl

    8 mM Na2HPO4

    2 mM KH2PO4

    Filter sterilize.

  5. PBS dialysis buffer (pH 7.4)

    137 mM NaCl

    2.7 mM KCl

    8 mM Na2HPO4

    2 mM KH2PO4

    5% glycerol

    Autoclave and add 5 mM 2-mercaptoethanol immediately prior to use.

Acknowledgments

This work was supported by a National Research Foundation (NRF) of Korea grant funded by the Korean government (NRF-2017R1A2B3009624 to J.H.A.) and Samsung Science and Technology Foundation (SSTF-BA1602-12 to J.H.A). The authors thank H.K. Song for fluorescence polarization spectroscopy experiments.

Competing interests

The authors declare no competing interests.

References

  1. Bernier, I., Tresca, J. P. and Jolles, P. (1986). Ligand-binding studies with a 23 kDa protein purified from bovine brain cytosol. Biochim Biophys Acta 871(1): 19-23.
  2. Jin, S., Nasim, Z., Susila, H. and Ahn, J. H. (2021). Evolution and functional diversification of FLOWERING LOCUS T/TERMINAL FLOWER 1 family genes in plants. Semin Cell Dev Biol 109: 20-30.
  3. Kim, S. C., Nusinow, D. A., Sorkin, M. L., Pruneda-Paz, J. and Wang, X. (2019). Interaction and Regulation Between Lipid Mediator Phosphatidic Acid and Circadian Clock Regulators. Plant Cell 31(2): 399-416.
  4. Nakamura, Y., Andres, F., Kanehara, K., Liu, Y. C., Dormann, P. and Coupland, G. (2014). Arabidopsis florigen FT binds to diurnally oscillating phospholipids that accelerate flowering. Nat Commun 5: 3553.
  5. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., et al. (2012). Fiji: an open-source platform for biological-image analysis. Nature Methods 9(7): 676-682.
  6. Susila, H., Juric, S., Liu, L., Gawarecka, K., Chung, K. S., Jin, S., Kim, S. J., Nasim, Z., Youn, G., Suh, M. C., Yu, H. and Ahn, J. H. (2021). Florigen sequestration in cellular membranes modulates temperature-responsive flowering. Science 373(6559): 1137-1142.
  7. Yao, H., Wang, G., Guo, L. and Wang, X. (2013). Phosphatidic acid interacts with a MYB transcription factor and regulates its nuclear localization and function in Arabidopsis. Plant Cell 25(12): 5030-5042.

简介


[摘要]蛋白质-脂质相互作用在许多生物过程中发挥着重要作用,包括代谢、信号传导和运输;然而,计算和结构分析通常无法预测这种相互作用,并且确定哪些脂质参与这些相互作用仍然具有挑战性。用于评估感兴趣的蛋白质和一组磷脂之间的物理相互作用的体外测定为预测这些相互作用在体内的功能提供了重要信息。在我们在评估拟南芥FLOWERING LOCUS T 的蛋白质-脂质结合的背景下开发的本方案中,我们描述了四个独立的体外实验,以确定蛋白质与磷脂的相互作用:脂质-蛋白质覆盖测定、脂质体结合化验、生物素-磷脂下拉化验和荧光偏振化验。这些互补的测定使研究人员能够测试感兴趣的蛋白质是否与测试组中的脂质相互作用,识别相关的脂质,并评估相互作用的强度。


[背景]蛋白质-脂质相互作用是许多细胞功能的基础。特别是,涉及质膜和其他细胞膜中脂质的相互作用在运输、信号传导和代谢中具有重要作用。蛋白质还与脂质生物合成中的脂质相互作用。此外,脂质分解代谢为动物和许多植物的种子发芽提供能量。尽管蛋白质-脂质相互作用很重要,但蛋白质序列和结构分析无法预测许多此类相互作用(Yao等人,2013;Kim等人,2019)。此外,识别这些相互作用中涉及的特定脂质仍然具有挑战性。
除了它们在其他过程中众所周知的作用外,蛋白质-磷脂相互作用还能调节拟南芥的开花时间(Nakamura等人,2014;Susila等人,2021)。在此过程中,FLOWERING LOCUS T (FT) 蛋白与磷脂酰甘油 (PG) 特异性相互作用并被隔离在膜中以防止早熟开花,尤其是在低环境温度下 (Susila et al. , 2021)。 FT 属于磷脂酰乙醇胺结合蛋白 (PEBP) 家族,并具有一个保守的阴离子结合口袋,可负责结合 PG (Bernier et al. , 1986; Jin et al. , 2021)。脂质结合也会影响其他调节因素;例如,一些没有跨膜结构域的转录因子与磷脂酸 (PA) 相互作用以调节植物发育和胁迫反应 (Yao et al. , 2013; Kim et al. , 2019)。对磷脂结合蛋白的研究可能会揭示复杂生物系统中的其他新调控机制。
在这里,我们描述了四种用于筛选 FT 与磷脂相互作用的方法:1)脂质-蛋白质覆盖测定,2)脂质体结合测定,3)生物素-磷脂下拉测定,和 4)荧光偏振测定(Susila等人,2021)。我们认为,有必要使用不同的体外方法来验证蛋白质-磷脂的相互作用。这种多功能方案可用于任何感兴趣的蛋白质,但应测试每种蛋白质的孵育温度和孵育持续时间。这些测定提供了关于蛋白质-脂质相互作用的补充信息,包括感兴趣的蛋白质是否与测试组中的脂质相互作用、它喜欢哪种脂质以及它与每种脂质相互作用的强度。

关键字:蛋白质-磷脂相互作用, 体外试验, FLOWERING LOCUS T, 磷脂酰甘油, 磷脂酰胆碱, 磷脂, 脂质体, 光偏振

材料和试剂
A.材料
按照标准方法从大肠杆菌中纯化重组 FT 蛋白( Susila et al. , 2021 )。磷脂和半乳糖脂获自 Sigma-Aldrich 或 Avanti Polar Lipids(表 1)。


表 1. 本研究中使用的脂质
脂质缩写小贩目录编号
1,2-二棕榈酰-sn-甘油-3-磷酸DPPAAvanti 极性脂质830855P
1,2-二棕榈酰-sn -glycero-3-phospho-1'-myo-inositol)DPPIAvanti 极性脂质850141P
1,2-二棕榈酰-sn -glycero-3-phospho-1'-rac-glycerol)DPPGAvanti 极性脂质840455P
1,2-二棕榈酰-sn-甘油-3-磷酸-L-丝氨酸DPPSAvanti 极性脂质840037P
1,2-二棕榈酰-sn-甘油-3-磷酸胆碱DPPCAvanti 极性脂质850355
1,2-二棕榈酰-sn-甘油-3-磷酸乙醇胺DPPEAvanti 极性脂质850705
来自蛋黄卵磷脂的L-α-磷脂酰-DL-甘油铵盐PGSigma-AldrichP0514
来自蛋黄的 L-α-磷脂酰乙醇胺体育Sigma-AldrichP7943
1-oleoyl-2-[12-biotinyl(aminododecanoyl)]- sn -glycero -3- phospho-(1'- rac -glycerol )(钠盐)生物素-PGAvanti 极性脂质860581C
1-oleoyl-2-[12-biotinyl(aminododecanoyl)]- sn -glycero -3- phosphocholinet生物素-PCAvanti 极性脂质860563C


B.脂质-蛋白质覆盖测定
1.Volac巴斯德吸管(Sigma-Aldrich,目录号:Z310727)
2.聚偏二氟乙烯(PVDF)膜(Millipore,目录号:IPVH304F0)
3.X射线胶片(AGFA,目录号:CP-BU New)


4.氯仿(Merck,目录号:1.02445.1000)
5.不含脂肪酸的牛血清白蛋白(Sigma-Aldrich,目录号:A6003)
6.抗 His单克隆抗体( Sigma-Aldrich,目录号: H1029;1:2,000稀释)
7.抗 Strep-tag II 单克隆抗体(Abcam,目录号: ab76949;1:2,000 稀释度)
8.山羊抗小鼠 IgG(Thermo Fisher Scientific,目录号: A28177; 1:10,000 稀释)
9.Western Blotting Detection Reagent Kit Ab Signal ( AbClon ,目录号: ABC-3001)
10.Tris( Duchefa ,目录号:T1501)
11.NaCl( Duchefa ,目录号:S0520)
12.甘油( Duchefa ,目录号:G1345)
13.2-巯基乙醇(Sigma-Aldrich,目录号:M6250)
14.纯化的标记蛋白(透析缓冲液中的 His-FT 或 FT- StII ;见配方)
15.Tris 缓冲盐水 (TBS)(见配方s )


C.脂质体结合试验
1.Volac巴斯德吸管(Sigma-Aldrich,目录号:Z310727)
2.蛋白质LoBind管(Eppendorf,目录号:0030108116)
3.PVDF膜(Millipore,目录号:IPVH304F0)
4.X射线胶片(AGFA,目录号:CP-BU New)
5.氯仿(Merck,目录号:1.02445.1000)
6.脱脂牛奶(Sigma-Aldrich,目录号:70166)
7.牛血清白蛋白(MP Biomedicals,目录号:0216006980)
8.抗 His单克隆抗体( Sigma-Aldrich,目录号: H1029;1:2,000稀释)
9.山羊抗小鼠 IgG(Thermo Fisher Scientific,目录号: A28177; 1:10,000 稀释)
10.Western印迹检测试剂盒Ab Signal( AbClon ,目录号: ABC-3001)
11.SDS(Sigma-Aldrich,目录号:L5750)
12.溴酚蓝(Sigma-Aldrich,目录号:B0126)
13.纯化的标记蛋白(透析缓冲液中的 His-FT 或 FT- StII ;见配方)
14.Tris 缓冲盐水 (TBS)(参见食谱)
15.SDS 样品缓冲液(参见配方)


D.生物素-磷脂下拉试验
1.Volac巴斯德吸管(Sigma-Aldrich,目录号:Z310727)
2.蛋白质LoBind管(Eppendorf,目录号:0030108116)
3.PVDF膜(Millipore,目录号:IPVH304F0)
4.X射线胶片(AGFA,目录号:CP-BU New)
5.氯仿(Merck,目录号:1.02445.1000)
6.乙醇(Merck,目录号:1.00983.1011)
7.Dynabeads MyOne Streptavidin T1(Invitrogen,目录号: 65601)
8.脱脂牛奶(Sigma-Aldrich,目录号:70166)
9.牛血清白蛋白(MP Biomedicals,目录号:0216006980)
10.抗 His单克隆抗体( Sigma-Aldrich,目录号: H1029;1:2,000稀释)
11.山羊抗小鼠 IgG(Thermo Fisher Scientific,目录号: A28177; 1:10,000 稀释)
12.Western印迹检测试剂盒Ab Signal( AbClon ,目录号: ABC-3001)
13.SDS 样品缓冲液(参见配方)
14.纯化的标记蛋白(透析缓冲液中的 His-FT;参见食谱)
15.Tris 缓冲盐水 (TBS)(参见食谱)


E.荧光偏振测定
1.Volac巴斯德吸管(Sigma-Aldrich,目录号:Z310727)
2.蛋白质LoBind管(Eppendorf,目录号:0030108116)
3.96孔黑色平底聚苯乙烯板(Corning,目录号:3915)
4.Pierce TM NHS-罗丹明抗体标记试剂盒(ThermoFisher Scientific,目录号:53031)
5.氯仿(Merck,目录号:1.02445.1000)
6.KCl (Sigma Aldrich,目录号:P3911)
7.Na 2 HPO 4 (Sigma-Aldrich,目录号:S9763)
8.KH 2 PO 4 (Sigma-Aldrich,目录号:P0662
9.纯化的标记蛋白(PBS 透析缓冲液中的 His-FT;参见食谱)
10.磷酸盐缓冲盐水 (PBS)(参见食谱)
11.PBS 洗脱缓冲液(参见配方)


设备


A.脂质-蛋白质覆盖测定
1.琥珀色玻璃小瓶(Shimadzu Scientific Korea,目录号:91005-0920)
2.涡流混合器(Scientific Industries,Genie 2,型号: G-560)
3.微量移液器(Eppendorf Research,目录号:3123000918)
4.蛋白质印迹托盘
5.轨道摇床(最佳实验室设备,型号:RF300)
6.X 光胶片暗盒
7.X 射线胶片处理器(Healthcare Solutions Inc.,型号:JP-33)


B.脂质体结合试验
1.琥珀色玻璃小瓶(Shimadzu Scientific Korea,目录号:91005-0920)
2.涡流混合器(Scientific Industries,Genie 2,型号:G-560)
3.微量移液器(Eppendorf Research,目录号:3123000918)
4.Bioruptor超声装置(Bio-Medical Science,型号:KRB 01)
5.冷冻离心机(Eppendorf Research,型号:5430R)
6.培养箱(Vision Scientific,型号:VS-8480MX2-DT)
7.加热块(最佳实验室设备,型号:CF1)
8.蛋白质印迹托盘
9.轨道摇床(最佳实验室设备,型号:RF300)
10.X 光胶片暗盒
11.X 射线胶片处理器(Healthcare Solutions Inc.,型号:JP-33)


C.生物素-磷脂下拉试验
1.琥珀色玻璃小瓶(Shimadzu Scientific Korea,目录号:91005-0920)
2.涡流混合器(Scientific Industries,Genie 2,型号:G-560)
3.微量移液器(Eppendorf Research,目录号:3123000918)
4.管旋转器( Seoulin Bioscience,型号:SLRM-3)
5.磁选架
6.降速离心机( Allsheng ,型号:mini-6K)
7.加热块(最佳实验室设备,型号:CF1)
8.蛋白质印迹托盘
9.轨道摇床(最佳实验室设备,型号:RF300)
10.X 光胶片暗盒
11.X 射线胶片处理器(Healthcare Solutions Inc.,型号:JP-33)


D.荧光偏振测定
1.琥珀色玻璃小瓶(Shimadzu Scientific Korea,目录号:91005-0920)
2.涡流混合器(Scientific Industries,Genie 2,型号:G-560)
3.微量移液器(Eppendorf Research,目录号:3123000918)
4.Bioruptor超声装置(Bio-Medical Science,型号:KRB 01)
5.冷冻离心机(Eppendorf Research,型号:5430R)
6.培养箱(Vision Scientific,型号:VS-8480MX2-DT)
7.SpectraMax多模式读板机(Molecular Devices)


软件


1.ImageJ Fiji,一个开源软件( Schindelin等人,2012 年)
2.GraphPad Prism 5(Graph Pad Software Inc.)


程序


A.脂质-蛋白质覆盖测定
在这种定性分析中,脂质被固定在膜上并与标记的蛋白质一起孵育。然后通过针对表位标签的抗体检测任何结合的蛋白质(图 1 和图 5A)。我们建议使用标签蛋白(例如,GST、GFP 或 MBP)或没有脂质结合能力的蛋白作为实验的阴性对照。


 
图 1. 脂质-蛋白质叠加分析。
脂质被固定在 PVDF 膜上并与感兴趣的蛋白质一起孵育。用蛋白质印迹检测脂质结合的蛋白质。


1.将脂质溶解在纯氯仿中以制备储备溶液 (1 mg/mL)。使用Volac Pasteur 移液器混合和转移脂质溶液。
2.将脂质储备溶液储存在 -20°C 的琥珀色玻璃瓶中。
3.切割 PVDF 膜(2 × 8 厘米)并标记脂质的斑点(至少相距 1 厘米)。
4.μg 的每种脂质(5 μL 库存溶液)缓慢点到准备好的 PVDF 膜(直径至少 1–2 毫米)上,并在室温下将膜干燥至少 30 分钟至 1 小时。
5.将膜置于蛋白质印迹托盘 (6 × 10 cm) 中,用 10 mL 封闭溶液(含有3% [w/v] 无脂肪酸牛血清白蛋白的TBS )在室温下温和孵育1 小时搅拌(10 转/分)。
6.弃去封闭溶液,将膜与 5 mL 封闭溶液中的 60 µg 重组 His-FT 或 FT- StII蛋白一起在 4°C 温和搅拌(10 rpm)孵育 16 小时。
7.弃去蛋白质溶液,用 10 mL TBS 洗涤膜 5 分钟;总共重复洗涤四次。
8.将膜与含有一抗(抗 His 单克隆抗体或抗 Strep-tag II 单克隆抗体)的 10 mL 封闭溶液在室温下轻轻搅拌(10 rpm)孵育 1 小时。
9.弃去抗体溶液,用 10 mL TBS 洗膜 5 分钟;重复洗涤四次。
10.用含有山羊抗小鼠 IgG 二级抗体的 10 mL 封闭溶液在室温下孵育膜 1 小时,并轻轻搅拌(10 rpm)。
11.用 10 mL TBS 溶液清洗膜 5分钟;重复洗涤四次。
12.用 2 mL 的辣根过氧化物酶 (HRP) 底物溶液孵育膜 5 分钟。
13.去除膜上多余的液体,将用塑料包裹的膜放入 X 射线胶片暗盒中,并使用带有 X 射线胶片处理器的 X 射线胶片检测信号。


B.脂质体结合试验
在这种定性分析中,标记的蛋白质与由两种脂质的不同比例组成的脂质体一起孵育;结合蛋白被脂质体拉下并通过蛋白质印迹检测(图 2 和图 5B)。我们建议读者使用标签蛋白(例如,GST、GFP 或 MBP)或没有脂质结合能力的蛋白作为实验的阴性对照。


 
图 2. 脂质体结合试验。
将感兴趣的蛋白质与由不同比例的两种脂质组成的脂质体一起孵育。通过蛋白质印迹沉淀和检测脂质体结合的蛋白质。


1.将脂质溶解在纯氯仿中以制备储备溶液 (1 mg/mL)。使用Volac Pasteur 移液器混合和转移脂质溶液。
2.将脂质储备溶液储存在 -20°C 的琥珀色玻璃瓶中。
3.为了制造脂质体,将磷脂溶液以所需比例转移到蛋白质LoBind管中,每次反应 50 µg脂质。例如,PG/PC 脂质体:50/0 µg、40/10 µg、30/20 µg、20/30 µg、10/40 µg 和 0/50 µg。
4.打开管子蒸发氯仿,在每个管子中获得脂质膜。
5.µL TBS对脂质膜进行再水化,并在37°C 下孵育 1 小时。
6.涡旋以分散脂质 10 分钟。
7.在冰水浴中对脂质混合物(高设置:320W)进行声波处理 10 分钟(30秒开启和 30 秒关闭)。
8.更换水浴中的冰水并重复步骤B7。
9.g和 4°C 下将脂质体离心 10 分钟。
10.丢弃上清液,用 100 μL的冷 TBS清洗脂质体两次。
11.将脂质体重新悬浮在 50 μL 的冷 TBS 中(最终浓度为 1 mg/mL)。
12.将 1 µg 纯化蛋白 (His-FT) 添加到脂质体溶液中,并在 30°C 下轻轻搅拌孵育 30 分钟。使用一管不含脂质体(仅 TBS)作为阴性对照。
13.g和 4°C 下将混合物离心 10 分钟。
14.取出上清液并储存在另一个试管中以进行蛋白质印迹控制。
15.μL的冷 TBS 溶液清洗脂质体两次。涡旋混合物,直到所有颗粒完全重新悬浮。
16.将 10 µL的 6× SDS 样品缓冲液添加到步骤B1 3 的沉淀和上清液中,并在 95°C 的加热块中孵育样品5 分钟。将样品储存在-20°C 直至进一步使用。
17.为 SDS-PAGE 准备 12% 标准聚丙烯酰胺凝胶(迷你凝胶:10 × 8 厘米;11 口井)。
18.将样品装入井中,用 110 V 运行凝胶 2 小时,然后将蛋白质转移到 PVDF 膜上。
19.将膜放入西方印迹托盘中,用 10 mL 的阻塞溶液(含有3% [w/v] 脱脂牛奶的 TBS)在室温下搅拌 1 小时(50 rpm)孵育。
20.2 O短暂清洗膜。
21.,将膜与一抗(抗 His 单克隆抗体)在 10 mL 含有 1% (w/v) 牛血清白蛋白的 TBS 中孵育过夜。
22.用 10 mL TBS 溶液清洗膜 5分钟四次。
23.在室温下用山羊抗鼠 IgG 二级抗体在含有 1% (w/v) 牛血清白蛋白的 10 mL TBS 中孵育膜 1 小时。
24.用 10 mL TBS 溶液清洗膜 5分钟四次。
25.用 2 mL 的 HRP 底物溶液孵育膜 5 分钟。
26.去除膜上多余的液体,将用塑料包裹的膜放入 X 射线胶片暗盒中,并使用带有 X 射线胶片处理器的 X 射线胶片检测信号。


C.生物素-磷脂下拉试验
在这种定性分析中,生物素化的脂质与蛋白质一起孵育;用链霉亲和素珠子将脂质拉下,并通过蛋白质印迹检测结合蛋白(图 3 和图 5C)。




 
图 3. 生物素-磷脂下拉试验。
生物素标记的磷脂与感兴趣的蛋白质一起孵育。蛋白质-脂质复合物被链霉亲和素珠拉下。通过蛋白质印迹检测脂质结合的蛋白质。


1.将生物素标记的脂质溶解在纯氯仿中以制备库存溶液 (1 mg/mL)。使用Volac Pasteur 移液器混合和转移脂质溶液。
2.将脂质储备溶液储存在 -20°C 的琥珀色玻璃瓶中。
3.将 15 µL (15 µg)的每种生物素标记的磷脂转移到蛋白质LoBind中 管,蒸发氯仿,得到脂质膜。
4.μL 的 50% 乙醇补充脂质膜。
5.在室温下孵育脂质溶液 15 分钟,每 5 分钟短暂涡旋一次。
6.加入 40 μL 的含有1 μg 纯化蛋白的 TBS。
7.1 μg 纯化蛋白(不含脂质溶液)的50 μL TBS作为阴性对照。
8.孵育30 分钟,同时轻轻搅拌。
9.在步骤 C7 的孵育过程中,通过转移 20 μL Dynabeads来制备珠子 MyOne链霉亲和素 T1 到蛋白质LoBind管。
10.架收集珠子,用含有 0.5%(w/v)牛血清白蛋白的 500 μL 的 TBS 洗涤珠子两次。
11.含有 0.5% (w/v) 牛血清白蛋白的 TBS 的 450 μL 中重新悬浮珠子。
12.将步骤 C7 中的脂质-蛋白质混合物短暂离心,将 5 µL 上清液放入新试管中作为输入对照(10% 输入),然后将其余混合物转移到含有珠子的试管中。将输入样品储存在 -20°C。
13.将珠-脂质-蛋白质混合物在试管旋转器 (10 rpm) 中在4°C 下孵育过夜。
14.用磁性分离架收集珠子,用含有 0.5%(w/v)牛血清白蛋白的 500 μL 的 TBS 清洗珠子两次。
15.从最后一个洗涤步骤收集珠子,并将 SDS 样品缓冲液添加到珠子和来自步骤 C11 的输入样品中。在加热块中将样品在 95°C 下孵育5 分钟。将样品储存在-20°C。
16.为 SDS-PAGE 准备标准的 12% 聚丙烯酰胺凝胶。
17.将样品装入井中,用 110 V 运行凝胶 2 小时,然后将蛋白质转移到 PVDF 膜上。
18.将膜放入西方印迹托盘中,在室温下用 10 mL 的阻塞溶液(含有3% [w/v] 脱脂牛奶的 TBS)孵育1 小时,并轻轻搅拌(50 rpm)。
19.2 O短暂清洗膜。
20.将膜与一抗(抗 His 单克隆抗体)在 10 mL 含有 1% (w/v) 牛血清白蛋白的 TBS 中于4°C 孵育过夜。
21.弃去抗体溶液,用 10 mL TBS 洗膜 5 分钟;重复洗涤四次。
22.在室温下用山羊抗鼠 IgG 二级抗体在含有 1% (w/v) 牛血清白蛋白的 10 mL TBS 中孵育膜 1 小时。
23.用 10 mL TBS 溶液清洗膜 5分钟;重复洗涤四次。
24.用 2 mL 的 HRP 底物溶液孵育膜 5 分钟。
25.去除膜上多余的液体,将用塑料包裹的膜放入 X 射线胶片暗盒中,并使用带有 X 射线胶片处理器的 X 射线胶片检测信号。


D.荧光偏振测定
在这种定量分析中,蛋白质-脂质相互作用会改变荧光团发出的光的极性,该荧光团附着在感兴趣的蛋白质上并被偏振光激发;将所得的各向异性值绘制为脂质体浓度的函数图给出了蛋白质-脂质结合亲和力(图 4 和图 5D)。


 
图 4. 荧光偏振测定。
荧光染料标记的蛋白质与脂质体一起孵育。通过荧光偏振或各向异性的值计算相互作用的程度。


1.将脂质溶解在纯氯仿中以制备储备溶液 (1 mg/mL)。使用Volac Pasteur 移液器混合和转移脂质溶液。
2.将脂质储备溶液储存在 -20°C 的琥珀色玻璃瓶中。
3.μL的 PBS 透析缓冲液中制备 1 mg 纯化蛋白(His-FT) 。避免在缓冲液中使用任何胺分子,例如 Tris 或甘氨酸。
4.要标记蛋白质,请将 500 μL 的蛋白质溶液添加到包含试剂盒中的 NHS-罗丹明试剂的小瓶中,并与移液器混合,直到所有染料溶解。
5.在黑暗中在室温下孵育小瓶 1 小时。
6.要去除未结合的染料,请通过涡旋和移液从试剂盒中重新悬浮树脂来制备两个自旋柱,然后将 400 μL 的树脂转移到每个自旋柱上。
7.在室温下以1,000 × g将柱子离心 30 秒以去除缓冲液。
8.将色谱柱放入新的收集管中,将步骤D5中的 250 µL 蛋白质-染料混合物添加到离心柱中。
9.通过涡旋和移液混合样品。
10.在室温下以 1,000 × g将色谱柱离心 30 秒。
11.收集并合并来自两个收集管的流出液。
12.用 PBS 透析缓冲液将标记的蛋白质混合物稀释至4 μM的浓度 (相当于 His-FT 的浓度为0.1 µg/µL)作为库存。
13.将标记的蛋白质分装并储存在 -20°C 的蛋白质LoBind管中,避光保存,直至进一步使用。
14.为了制造脂质体,将磷脂溶液转移到微量离心管中以产生所需的浓度系列,如下所述。在为系列稀释分析制作储备溶液之前计算所需的磷脂量。详细信息,包括磷脂的分子量,可从 Avanti Polar Lipids 网站 ( https://avantilipids.com/ ) 获得。
在这种情况下,我们使用 DPPG (MW: 744.492 g/mol) 和 DPPC (MW: 733.562 g/mol)。我们在 200 µL 反应体积中使用浓度为 0、10、20、30、50、75、100和 150 µM 的脂质连续稀释,并进行了三个技术重复(来自 2 mM 储备溶液)。因此,我们将 298 μL 的 DPPG 和 293 μL 的 DPPC 储备溶液 ( 1 mg/mL )转移到微量离心管中,以制备 200 μL 的 2 mM 储备脂质体。
15.蒸发氯仿以获得脂质膜。
16.µL 的 PBS再水化脂质膜,并在37°C 下孵育 1 小时。
17.涡旋脂质分散 10 分钟。
18.在冰水浴中对样品(高设置)进行两次 10 分钟(30秒开和 30 秒关)。
19.g和 4°C 下将脂质体离心 10 分钟。
20.μL的冷 PBS清洗脂质体两次。
21.将脂质体重新悬浮在 200 μL 的冷 PBS 中(最终浓度为 2 mM)。
22.准备 96 孔板并将 1.5 μL的蛋白质、脂质体和 PBS 转移到每个孔中。将总体积设置为 200 μL(表 1)。如上所述,将脂质体稀释至 0、10、20、30、50、75、100 和 150 µM 的浓度。

表 1. 测定中各组分的组成
零件脂质体 
(0 µM)脂质体
(10 µM)脂质体
(20 µM)-----脂质体
(150 µM)
蛋白质 ( µL)
脂质体 (µL)
PBS (µL)1.5 µL
0 µL
198.5 µL1.5 µL
1 µL
197.5 µL1.5 µL
2 µL
196.5 µL-----1.5 µL
15 µL
183.5 µL


23.将板在室温下孵育 30 分钟,轻轻搅拌。
24.准备SpectraMax多模式读板机。在软件界面中,将温度设置为30°C,选择endpoint measurement with top read,选择Costar black 96-well with no lid作为检测板,选择auto mix for 3 s。最后,将激发波长设置为 544 nm,发射波长设置为 575 nm,截止设置为 570 nm。
25.使用该软件计算各向异性 ( r ) 值。这些值可以保存到文本 (.txt) 文件或手动记录。
26.将获得的数据(一式三份)绘制到 GraphPad Prism 5 软件中的散点图中,x 轴上的脂质体浓度和 y 轴上的各向异性 ( r ) 值。
27.使用非线性回归模型(一个站点,总绑定)分析数据以获得解离常数以确定结合亲和力。


数据分析


代表性数据


 
图 5.体外FT 蛋白-磷脂相互作用测定的代表性结果。 
A.脂质-蛋白质覆盖测定。 B.脂质体结合测定。 C.生物素-磷脂下拉测定。 D.荧光偏振测定。 DPPI:二棕榈酰磷脂酰肌醇,DPPA:二棕榈酰磷脂酸,DPPS:二棕榈酰磷脂酰丝氨酸,DPPG:二棕榈酰磷脂酰甘油,DPPE:二棕榈酰磷脂酰乙醇胺,DPPC:二棕榈酰磷脂酰胆碱,PG:磷脂酰甘油,PE:磷脂酰乙醇胺。
食谱


以下配方中使用的水是双蒸水 (ddH 2 O)。
1.透析缓冲液(pH 8.0)
50 毫米三
150 毫米氯化钠
5% 甘油
过滤灭菌并在使用前立即添加 5 mM 2-巯基乙醇。
2.Tris 缓冲盐水 (TBS) (pH 7.2)
50 毫米三
150 毫米氯化钠
过滤除菌。
3.4× SDS 样品缓冲液
200 mM Tris-Cl pH 6.8
8% 安全数据表
0.4% 溴酚蓝
40% 甘油
4.磷酸盐缓冲液 (PBS) (pH 7.4)
137 毫米氯化钠
2.7 毫米氯化钾
8 毫米钠2 HPO 4
2 毫米 KH 2 PO 4
过滤除菌。
5.PBS 透析缓冲液 (pH 7.4)
137 毫米氯化钠
2.7 毫米氯化钾
8 毫米钠2 HPO 4
2 毫米 KH 2 PO 4
5% 甘油
高压灭菌器并在使用前立即添加 5 mM 2-巯基乙醇。


致谢


这项工作得到了韩国政府 (NRF-2017R1A2B3009624 至 JHA) 和三星科技基金会 (SSTF-BA1602-12 至 JHA) 资助的韩国国家研究基金会 (NRF) 的支持。作者感谢 HK Song 的荧光偏振光谱实验。




利益争夺


作者声明没有竞争利益。


参考


1.Bernier, I.、 Tresca , JP 和Jolles , P. (1986)。用从牛脑细胞质中纯化的 23 kDa 蛋白质进行的配体结合研究。 生物化学 生物物理学报871(1):19-23。
2.Jin , S.、Nasim, Z.、Susila, H. 和Ahn , JH (2021)。植物中FLOWERING LOCUS T/TERMINAL FLOWER 1家族基因的进化和功能多样化。 精细胞开发生物学109:20-30。
3.Kim, SC, Nusinow , DA, Sorkin, ML, Pruneda -Paz, J. 和 Wang, X. (2019)。脂质介质磷脂酸和生物钟调节剂之间的相互作用和调节。 植物细胞31(2):399-416。
4.Nakamura, Y.、Andres, F.、Kanehara, K.、Liu, YC、 Dormann , P. 和 Coupland, G. (2014)。拟南芥FT 与昼夜振荡的磷脂结合,可加速开花。 国家通讯5:3553 。
5.Schindelin , J., Arganda -Carreras, I., Frise, E., Kaynig , V., Longair , M., Pietzsch , T., Preibisch , S., Rueden , C., Saalfeld, S., Schmid, B .,等人。 (2012)。斐济:一个用于生物图像分析的开源平台。 自然方法9(7):676-682。
6.Susila, H., Juric , S., Liu, L., Gawarecka , K., Chung, KS, Jin , S., Kim, SJ, Nasim, Z., Youn , G., Suh, MC, Yu, H . 和Ahn ,JH(2021 年)。细胞膜中的 Florigen 隔离调节温度响应性开花。 科学373(6559):1137-1142。
7.Yao, H.、Wang, G.、Guo, L. 和 Wang, X. (2013)。磷脂酸与 MYB 转录因子相互作用并调节其在拟南芥中的核定位和功能。 植物细胞25(12):5030-5042。

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Copyright: © 2022 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. Susila, H., Jurić, S., Gawarecka, K., Chung, K. S., Jin, S., Kim, S. J., Nasim, Z., Youn, G. and Ahn, J. H. (2022). In vitro Assays to Evaluate Specificity and Affinity in Protein-phospholipid Interactions. Bio-protocol 12(10): e4421. DOI: 10.21769/BioProtoc.4421.
  2. Susila, H., Juric, S., Liu, L., Gawarecka, K., Chung, K. S., Jin, S., Kim, S. J., Nasim, Z., Youn, G., Suh, M. C., Yu, H. and Ahn, J. H. (2021). Florigen sequestration in cellular membranes modulates temperature-responsive flowering. Science 373(6559): 1137-1142.
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