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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

HS Hendry Susila
SJ Snježana Jurić
KG Katarzyna Gawarecka
KC Kyung Sook Chung
SJ Suhyun Jin
SK Soo-Jin Kim
ZN Zeeshan Nasim
GY Geummin Youn
JA Ji Hoon Ahn

发布: 2022年05月20日第12卷第10期 DOI: 10.21769/BioProtoc.4421 浏览次数: 2749

评审: Laxmi Narayan MishraNityanand SrivastavaSashi ReddyAnonymous reviewer(s)

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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) search In vitro assay (体外试验) search FLOWERING LOCUS T (FLOWERING LOCUS T) search Phosphatidylglycerol (磷脂酰甘油) search Phosphatidylcholine (磷脂酰胆碱) search Phospholipids (磷脂) search Liposome (脂质体) search Fluorescence polarization (光偏振) search

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

    LipidAbbreviationVendorCatalog number
    1,2-dipalmitoyl-sn-glycero-3-phosphateDPPAAvanti Polar Lipids830855P
    1,2-dipalmitoyl-sn-glycero-3-phospho-1'-myo-inositol)DPPIAvanti Polar Lipids850141P
    1,2-dipalmitoyl-sn-glycero-3-phospho-1'-rac-glycerol)DPPGAvanti Polar Lipids840455P
    1,2-dipalmitoyl-sn-glycero-3-phospho-L-serineDPPSAvanti Polar Lipids840037P
    1,2-dipalmitoyl-sn-glycero-3-phosphocholineDPPCAvanti Polar Lipids850355
    1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamineDPPEAvanti Polar Lipids850705
    L-α-phosphatidyl-DL-glycerol ammonium salt from egg yolk lecithinPGSigma-AldrichP0514
    L-α-phosphatidylethanolamine from egg yolkPESigma-AldrichP7943
    1-oleoyl-2-[12-biotinyl(aminododecanoyl)]-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)tBiotin-PGAvanti Polar Lipids860581C
    1-oleoyl-2-[12-biotinyl(aminododecanoyl)]-sn-glycero-3-phosphocholinetBiotin-PCAvanti Polar Lipids860563C


  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

English
中文翻译

文章信息

版权信息

© 2022 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

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.

    3. ris ris Download Citation in RIS Format

分类

植物科学 > 植物生物化学 > 蛋白质

生物化学 > 蛋白质 > 相互作用

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