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Jan 2019

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Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides
利用光敏阿米洛利光亲和标记牛心脏亚线粒体颗粒中呼吸链复合物I   

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Abstract

The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [125I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [125I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.

Keywords: Photoaffinity labeling (光亲和标记), Respiratory complex I (呼吸链复合物I), Amilorides (阿米洛利), Enzyme inhibitor (酶抑制剂), Mitochondria (线粒体), Bioenergetics (生物能量学), NADH-quinone oxidoreductase (NADH泛醌氧化还原酶), Chemical biology (化学生物学)

Background

Proton-translocating NADH-quinone oxidoreductase (respiratory complex I) is a multi-subunit membrane protein complex, which catalyzes the initial step of mitochondrial/bacterial electron transport chains (Hirst, 2013). The recent progress in X-ray crystallography and cryo-EM enabled modeling the entire structure of complex I. The structural studies proposed the quinone/inhibitor-access channel model to explain how quinone/inhibitor bind to the enzyme (Sazanov, 2015; Wirth et al., 2016). However, since there is currently no structural data of complex I with bound-quinone or inhibitor, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle.

Photoaffinity labeling technique, which uses a synthetic ligand that possesses photoreactive group such as diazirine and phenyl-azido, is the most commonly used method of affinity-based protein modification (Hatanaka and Sadakane, 2002). It provides a powerful means of investigating interactions between biologically active compounds and the proteins of interest. Recently, to get insights into the structure of quinone/inhibitor-binding site in mitochondrial complex I, we carried out photoaffinity labeling using photoreactive [125I]amilorides ([125I]PRA3, [125I]PRA4, [125I]PRA5, and [125I]PRA6) with bovine heart submitochondrial particles (SMPs) (Uno et al., 2019). The radioisotope (125I) was used as a tracer, which enables the experiment with the lowest concentrations of ligands (1-10 nM). These concentrations are approximately two orders of magnitude lower than those of alkyne-tagged amilorides (PRA1 and PRA2, Murai et al., 2015), which can be conjugated with biotin or fluorophores via Cu+-catalyzed click chemistry (Wang et al., 2003) after cross-linking reaction. This advantage may minimize the possibility of non-specific labeling, which is a primary cause of false-positive results.

We herein describe the detailed procedures of the photoaffinity labeling of complex I in bovine SMPs by [125I]PRA5 as an example (Figure 1), which includes 1) preparation of 125I-tagged photoreactive ligand, 2) cross-linking of bovine SMPs by the ligand, and 3) identification of the labeled protein(s).



Figure 1. Schematic presentation of photoaffinity labeling of bovine heart SMPs by [125I]amilorides

Materials and Reagents

  1. 1.5 ml Screw-capped microtube (Watson, catalog number: 139-115-112C)
  2. 1.5 ml microtube (Safe-Lock Tube, Eppendorf, catalog number: 0030120086)
  3. Pipette tips (Gilson)
  4. Membrane filter unit (Amicon-Ultra, 100 kDa, Merck Millipore, catalog number: UFC510096)
  5. Reverse-phase HPLC column (COSMOSIL 5C18-MSII, 4.6 mm x 200 mm, Nacalai Tesque) 
  6. TLC plate (Merck Millipore, catalog number: 1.05554.0001)
  7. Plastic wrap
  8. Amiloride-tin-precursor (A21a, 1 .0 mM in ethanol, Figure 1A, The synthetic procedures are described in Uno et al., 2019)
  9. [125I]NaI (carrier-free, 2,000 Ci/mmol, Perkin-Elmer, catalog number: NEZ033A)
  10. Chloramine T (Wako Pure Chemicals, catalog number: 032-02182)
  11. Sodium hydrogen sulfite (NaHSO3, Wako Pure Chemicals, catalog number: 198-01371)
  12. Bovine submitochondrial particles (SMPs)
    Notes:
    1. SMPs are inside-out vesicles of inner mitochondrial membrane.
    2. Mitochondria were isolated from bovine heart as described elsewhere (Smith, 1967).
    3. SMPs were prepared on the basis of the previously described protocol (Matsuno-Yagi and Hatefi, 1985) using a sonication medium containing 0.25 M sucrose, 1.0 mM potassium succinate, 1.5 mM ATP, 10 mM MgCl2, 10 mM MnCl2, and 10 mM Tris/HCl (pH 7.4), followed by ultracentrifugation. They are stored in a buffer containing 250 mM sucrose and 10 mM Tris-HCl (pH 7.4) at -80 °C until used.
  13. NativePAGETM Bis-Tris protein gels (4-16% precast gel, Thermo Fisher, catalog number: BN1004BOX)
  14. NativePAGETM Runnning buffer and Cathode buffer additive (Thermo Fisher, catalog numbers: BN2001 and BN2002, respectively)
  15. Serva Blue G (CBB G-250, Serva, catalog number: 35050.01)
  16. n-Dodecyl-D-β-maltoside (DDM, Dojin, catalog number: D316)
  17. Glycerol (Wako Pure Chemicals, catalog number: 075-00616)
  18. Ethanol (Wako Pure Chemicals, catalog number: 057-00456)
  19. Methanol (Wako Pure Chemicals, catalog number: 137-01823)
  20. Acetic acid (Wako Pure Chemicals, catalog number: 017-00251)
  21. Sucrose (Wako Pure Chemicals, catalog number: 192-00017)
  22. MgCl2 (hexahydrate, Wako Pure Chemicals, catalog number: 135-00165)
  23. NaCl (Wako Pure Chemicals, catalog number: 195-01665)
  24. Ponceau S (Nacalai Tesque, catalog number: 28322-72)
  25. Tricine (Dojin, catalog number: GB05)
  26. SDS (Wako Pure Chemicals, catalog number: 191-07145)
  27. Mercaptethanol (Wako Pure Chemicals, catalog number: 137-06862)
  28. Tris (Wako Pure Chemicals, catalog number: 011-20095)
  29. Acrylamide (Wako Pure Chemicals, catalog number: 011-08015)
  30. Bis-acrylamide (Wako Pure Chemicals, catalog number: 134-15081)
  31. Urea (Wako Pure Chemicals, catalog number: 219-00175)
  32. Silver Stain Kit (Wako Pure Chemicals, catalog number: 291-50301, compatible with MS)
  33. Trifluoroacetic acid (TFA)
  34. NADH (Oriental Yeast, catalog number: 44320000)
  35. Labeling buffer (see Recipes)
  36. 4x BN-PAGE sample buffer (see Recipes)
  37. Elution buffer (see Recipes)
  38. 4x Schägger sample buffer (see Recipes)
  39. 10x cathode buffer (see Recipes)
  40. 10x anode buffer (see Recipes)
  41. AB-3 mix (see Recipes)
  42. 3x gel buffer (see Recipes)
  43. 1.0 M KPi buffer, pH 7.4 (see Recipes)
  44. Gel composition for dSDS-PAGE (see Recipes)

Equipment

  1. Pippetmann (Gilson)
  2. -80 °C freezer (PHC, model: MDF-DU300H)
  3. HPLC system (Shimazdu, model: LC-10AS)
  4. Micro-centrifuge (CHIBITAN, Merck Millipore, model: XX42CF0RT)
  5. γ-counter (COBRATM II, Packard)
  6. Vacuum centrifugal evaporator (EYELA, model: CVE-2200)
  7. Bio-imaging analyzer (FLA-5100 or Typhoon FLA-9000 , Fuji Film or GE healthcare, respectively)
  8. Imaging plate (BAS IP MS 2040E, GE Healthcare)
  9. Vortex mixer (AS ONE, model: Trio TM-1N)
  10. Heat block (AS ONE, model: MyBL-10)
  11. Long wavelength UV lamp (UVP, Black-lay model B-100AP, equipped with a 100 W bulb, 365 nm)
  12. Electro-Eluter (Bio-Rad, model: 422)
  13. Gel dryer (Bio-Rad, Model583, equipped with HydroTech vacuum pump)
  14. Gel electrophoresis apparatus and glass plates (ATTO, AE-6500, 8 x 9 cm for mini-size gel format)

Software

  1. Image analysis software such as Multi Gauge or Image Quant (Fuji Film or GE Healthcare, respectively)

Procedure

  1. Preparation of photoreactive [125I]amilorides ([125I]PRA5 as an example) 
    1. Prepare ethanolic solution of Tin-precursor A21a (1.0 mM, 20 µl) in a screw-capped 1.5 ml microtube.
    2. Add [125I]NaI (1 mCi, 2,000 Ci/mmol, 10 μl).
      Note: [125I]NaI should be handled in a draft chamber until the reaction is quenched by NaHSO3.
    3. Initiate the reaction by the addition of aqueous chloramine T (3.0 mM in 1.0 M KPi buffer, pH 7.4, 10 µl).
      Note: Aqueous chloramine T solution should be freshly prepared for each experiment.
    4. Vortex and spin-down the tube.
    5. Incubate the mixture for 10 min at room temperature.
    6. Quench the reaction by the addition of 5% (w/v) aqueous sodium hydrogen sulfite (10 µl).
    7. Inject whole volume of the mixture (~50 µl) to the HPLC with a manual injection port (loop volume should be larger than 100 µl). LC conditions are as follows:
      1. Column: COSMOSIL 5C18-MSII, 4.6 mm x 200 mm (Nacalai Tesque)
      2. Solvent: methanol/0.01% aqueous TFA (step gradient elution of 1:9, then 9:1).
      3. Temperature: 35 °C
      4. Flow rate: 0.8 ml/min.
    8. Collect fractions every 30 s (~400 µl).
    9. Take 3 µl of the samples to assess the radioactivity and purity of each fractions by γ-counter (2 µl) and radio-TLC (1 µl), respectively (see Figure 2). 
    10. Combine the fractions containing [125I]PRA5 (frs. 8 and 9 in Figure 2), and concentrate the sample up to ~50 µl by a vacuum-centrifugal evaporator.
      Note: Do not dry the sample completely.
    11. Adjust the concentration of [125I]PRA5 to 1 mCi/ml by ethanol.
    12. Store the sample at 4 °C ([125I]amilorides are stable as ethanolic solutions for at least 2 months).


      Figure 2. Purification of photoreactive [125I]PRA5 by reverse phase HPLC. The mixture containing crude [125I]PRA5 was applied to a C18 column. The salts were removed by rinsing the column with 10% methanol/0.01% aqueous TFA (0-10 min), then [125I]PRA5 was eluted with an isocratic 90% methanol/0.01% aqueous TFA (10-20 min). The elute was fractionated every 30 s, and 2 µl aliquot from each fraction was transferred to RIA tube and the radioactivity was measured using γ-counter (retention time; 10-17.5 min). A. Radioactivity distribution in HPLC chromatogram. The radiochemical yield was approximately 70% from the initial [125I]NaI. B. TLC analysis of the collected fractions. The samples (1 µl) were analyzed on a TLC plate using 10% methanol/chloroform as a mobile phase. The plate was exposed on an imaging plate for 1 h and analyzed by Bio-imaging analyzed FLA-5100.

  2. Cross-linking of bovine SMPs by [125I]PRA5
    1. In a 1.5 ml microtube, add the suspension of bovine SMPs in the labeling buffer (2.0-4.0 mg/ml, 100-200 µl).
    2. Add [125I]PRA5 at the concentration of 5-10 nM.
      Note: Ethanol concentration should be below ~3%.
    3. Incubate at room temperature for 10 min.
    4. Add 50 µM NADH, then incubate for 5 min.
    5. Irradiate with a long wavelength UV lamp on ice for 10 min at the distance of 10 cm from the light source (Figure 3).
    6. Quench the cross-linking reaction by adding 10% (w/v) DDM to a final concentration 1.0%.
    7. Incubate the mixture on ice for 1 h.
    8. Add 4x BN-PAGE sample buffer.


      Figure 3. A set-up for photoaffinity labeling of bovine SMPs. The 1.5 ml Eppendorf tubes containing SMPs are placed with open lids on ice. They are UV-irradiated for 10 min at the distance of approximately 10 cm from the light source. For efficient labeling, the volume of the sample should be 100-200 µl/tube.

  3. Identification of the protein labeled by [125I]PRA5
    1. Isolation of complex I by Blue Native (BN)-PAGE and electroelution (Schägger, 2006)
      1. Add 5% Serva Blue G (CBB G-250). The CBB/DDM ratio should be adjusted to be 1/4 by weight. 
      2. Load samples into the 4-16% BN-precast gel (100-200 µg protein/well). 
      3. Start electrophoresis (100 V, 15 mA, for 1h). 
      4. Increase the voltage to 200 V (15 mA), and continue electrophoresis until the front dye reaches the bottom of the gel.
      5. Excise the complex I band with a razor blade.
        Note: Do not fix the BN gel. Do not include any unstained (or poorly stained) gel. Excise only the heart of the complex I band.
      6. Assemble the Electro-Eluter according to the manufacturer’s protocols.
      7. Fill the glass tube and the Eluter with elution buffer, and place the gel piece in the tube (4-5 gel pieces/tube).
      8. Elute protein at 10 mA/glass tube for overnight in a cold room.
        Note: If the elution is successful, the gel piece becomes colorless.
      9. Collect the eluted protein according to the manufacturer’s protocol (We recommend washing the dialysis membrane with 50-100 µl of 0.5% SDS to solubilize the precipitated proteins).
      10. Concentrate the collected solution with Amicon-Ultra 0.5 (100 kDa cut-off) to the appropriate volume (50-100 µl).
      11. Store the sample at -80 °C until used. 
    2. Resolution of complex I subunits by doubled SDS-PAGE (dSDS-PAGE, Rais et al., 2004; Wittig, 2006)
      1. Add 4x Schägger sample buffer to the complex I isolated by electroelution.
      2. Incubate the mixture at 40 °C for 1 h.
        Note: Do not boil the samples because some hydrophobic subunits in complex I aggregate.
      3. Separate complex I proteins on a first dimensional Schägger-type SDS gel (10% T, 3% C containing 6.0 M urea with 4% stacking gel, Table 1) with a voltage of 30 V (40 mA).
      4. When the samples have completely entered the gel, increase the voltage to 100 V, and continue electrophoresis until the dye front reaches the bottom of the gel.
      5. Excise a first dimensional gel strip and incubate it for 30 min in an acidic buffer containing 100 mM Tris/HCl (pH 2.0), then fix the strip between two glass plates (see Figures 4A, 4B, and 4C).
      6. Pour the second dimensional acrylamide mixture (16% T, 3% C, Table 1) and overlay with water (see Figure 4D).
      7. After polymerization, push down the first dimensional gel strip and make the two gels stick together (see Figure 4E). 
      8. Fill the gap with acrylamide mixture (16% T, 3% C, Table 1) containing 150 mM Tris/HCl (pH 7.4) and trace of Serva Blue G (spacer gel, see Figure 4F).
      9. Start second dimensional electrophoresis with a voltage of 30 V (40 mA).
      10. When the samples have completely entered the second dimensional gel, the voltage can be increased to 150 V. 
    3. Silver stain and autoradiography
      1. Fix and stain the gel with silver by an appropriate method (Yan et al., 2000) or by commercially available silver stain kit.
      2. Scan the silver-stained gel using a conventional flatbed scanner.
      3. Before place the gel on the gel drier, equilibrate the gel in a solution containing 3% glycerol, 40% methanol, and 10% acetic acid for 30 min.
      4. Dry the gel using gel drier at 65 °C for 3 h. Temperature should be raised slowly [about 1 °C/min (“Gradient mode”)].
      5. When the gel is completely dried, cover the gel with plastic wrap, then expose it onto the imaging plate for 12-24 h.
      6. Analyze the migration pattern of radio-labeled proteins by Bio-imaging analyzer, and compare those of silver-stained protein (representative results are shown in Figure 5).


        Figure 4. Preparation of the second dimensional gel for dSDS-PAGE. A. Excise the first dimensional gel strip. B. Carefully place the gel strip to a 15 ml falcon tube containing an acidic buffer. Incubate the gel for 30 min at room temperature. C. Fix the strip between two glass plates. D. Pour the second dimensional acrylamide mixture, and overlay with Milli-Q H2O. E. After polymerization of the acrylamide gel mixture, push down the gel strip to make the two gels stick together. F. Remove Milli-Q, then fill the gap with a spacer gel.


        Figure 5. Analysis of the proteins in SMPs labeled by [125I]PRA5. A. Separation of complex I by BN-PAGE. The SMPs labeled by [125I]PRA5 were separated on a 4-16% BN gel. The complex I band was identified by activity stain by NADH/NBT system, as described elsewhere (Murai et al., 2009; Shiraishi et al., 2012), and subjected to electroelution. Approximately 100 µg of SMPs proteins were loaded into each well. B. Resolution of complex I by dSDS-PAGE. The [125I]PRA5-labeled complex I, purified by BN-PAGE and electroelution, was separated on a first dimensional 10% Schägger-type Tricine gel (10% T, 3% C containing 6.0 M urea), followed by second dimensional separation on a 16% Schägger-type gel (16% T, 3% C). The 2D gel was subjected to silver stain (left) and autoradiography (right). The labeling by [125I]PRA5 provided two radioactive spots corresponding to the 49-kDa and PSST subunits, both of which comprise the quinone/inhibitor-binding pocket of complex I (Hirst 2013 and Sazanov 2015). The spots were identified by mass spectrometry and Western blotting (Shiraishi et al., 2012). We note that the weak radioactivity was found in a protein spot of ADP/ATP carrier (AAC), which was co-purified with complex I. The gel images are the same as those used in Figure 7C of the reference (Uno et al., 2019).

Recipes

  1. Labeling buffer (for UV irradiation of bovine SMP)
    250 mM sucrose
    50 mM KPi (pH 7.4)
    1 mM MgCl2
  2. 4x BN-PAGE sample buffer
    200 mM Bis-Tris/HCl (pH 7.2)
    200 mM NaCl
    40% (w/v) glycerol
    0.004% (w/v) Ponceau S
  3. Elution buffer (for electroelution of complex I)
    25 mM Tricine
    7.5 mM Bis-Tris/HCl (pH 7.0 adjusted at 4 °C)
  4. 4x Schägger sample buffer (for Tricine-PAGE)
    150 mM Tris/HCl (pH 7.0)
    12% (w/v) SDS
    30% (w/v) glycerol
    6% (w/v) mercaptoethanol
    0.05% (w/v) Serva Blue G
  5. 10x cathode buffer (for Schägger-type SDS-PAGE)
    1.0 M Tris
    1.0 M Tricine
    1% SDS
  6. 10x anode buffer (for Schägger-type SDS-PAGE)
    1.0 M Tris/HCl (pH 8.9) 
  7. AB-3 mix (49.5% T, 3% C, for Schägger-type SDS-PAGE)
    48% acrylamide
    1.5% bis-acrylamide
  8. 3x gel buffer (for Schägger-type SDS-PAGE)
    3.0 M Tris/HCl (pH 8.45)
    0.3% SDS
  9. 1.0 M KPi buffer, pH 7.4
    Dissolve K2HPO4 (12.1 g, 69.5 mmol) and KH2PO4 (4.1 g, 30.4 mmol) in 100 mL of distilled water.
  10. Gel composition for dSDS-PAGE (for Schägger-type SDS-PAGE) (Table 1)

    Table 1. Preparation of Schägger-type SDS gels (for two ATTO mini-size gels)

Acknowledgments

AcknowledgmentsThis protocol was adapted from Uno et al. (2019). This study was supported by JSPS KAKENHI (Grant Numbers JP26292060 and JP18H02147 to H.M., Grant Number JP18K05458 to M.M.). The experiments involving radioisotope techniques were performed at the Radioisotope Research Center, Kyoto University.

Competing interests

The authors declare that they have no conflicts of interest with the contents of this article.

References

  1. Hatanaka, Y. and Sadakane, Y. (2002). Photoaffinity labeling in drug discovery and developments: chemical gateway for entering proteomic frontier. Curr Top Med Chem 2(3): 271-288.
  2. Hirst, J. (2013). Mitochondrial complex I. Annu Rev Biochem 82: 551-575.
  3. Matsuno-Yagi, A. and Hatefi, Y. (1985). Studies on the mechanism of oxidative phosphorylation. Catalytic site cooperativity in ATP synthesis. J Biol Chem 260(27): 11424-11427.
  4. Murai, M., Sekiguchi, K., Nishioka, T. and Miyoshi, H. (2009). Characterization of the inhibitor binding site in mitochondrial NADH-ubiquinone oxidoreductase by photoaffinity labeling using a quinazoline-type inhibitor. Biochemistry 48(4): 688-698.
  5. Murai, M., Murakami, S., Ito, T. and Miyoshi, H. (2015). Amilorides bind to the quinone binding pocket of bovine mitochondrial complex I. Biochemistry 54(17): 2739-2746.
  6. Rais, I., Karas, M. and Schägger, H. (2004). Two-dimensional electrophoresis for the isolation of integral membrane proteins and mass spectrometric identification. Proteomics 4(9): 2567-2571. 
  7. Sazanov, L. A. (2015). A giant molecular proton pump: structure and mechanism of respiratory complex I. Nat Rev Mol Cell Biol 16(6): 375-388.
  8. Schägger, H. (2006). Tricine-SDS-PAGE. Nat Protoc 1(1): 16-22.
  9. Shiraishi, Y., Murai, M., Sakiyama, N., Ifuku, K. and Miyoshi, H. (2012). Fenpyroximate binds to the interface between PSST and 49 kDa subunits in mitochondrial NADH-ubiquinone oxidoreductase. Biochemistry 51(9): 1953-1963.
  10. Smith, A. L. (1967). Mitochondria : Slaughterhouse Material, Small-Scale. Methods Enzymol 10: 81-86.
  11. Uno, S., Kimura, H., Murai, M. and Miyoshi, H. (2019). Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches. J Biol Chem 294(2): 679-696.
  12. Wang, Q., Chan, T. R., Hilgraf, R., Fokin, V. V., Sharpless, K. B. and Finn, M. G. (2003). Bioconjugation by copper(I)-catalyzed azide-alkyne [3 + 2] cycloaddition. J Am Chem Soc 125(11): 3192-3193.
  13. Wirth, C., Brandt, U., Hunte, C. and Zickermann, V. (2016). Structure and function of mitochondrial complex I. Biochim Biophys Acta 1857(7): 902-914.
  14. Wittig, I., Braun, H. P. and Schägger, H. (2006). Blue native PAGE. Nat Protoc 1(1): 418-428.
  15. Yan, J. X., Wait, R., Berkelman, T., Harry, R. A., Westbrook, J. A., Wheeler, C. H. and Dunn, M. J. (2000). A modified silver staining protocol for visualization of proteins compatible with matrix‐assisted laser desorption/ionization and electrospray ionization‐mass spectrometry. Electrophoresis 21(17): 3666-3672.

简介

质子转运nadh醌氧化还原酶(呼吸复合物i)中醌/抑制剂通道的结构是用x射线晶体学和低温电镜模拟的,但通道模型是否反映了质子转运nadh醌氧化还原酶(呼吸复合物i)的生理相关态态仍有争议。催化循环。利用光活性的[125i]阿米洛利,我们证明阿米洛利类抑制剂与多个亚单位(49kda、nd1、psst和39kda亚单位)的界面区域结合,这很难与当前的通道模型相一致。本报告描述了用光活性[125i]阿米洛利标记牛亚软骨颗粒的方法。该方案可广泛应用于各种生物活性化合物的表征,其目标蛋白尚待鉴定或表征。
【背景】质子转运nadh醌氧化还原酶(呼吸复合物i)是一种多亚单位膜蛋白复合物,催化线粒体/细菌电子传递链的起始步骤(hirst,2013)。X射线晶体学和Cryo-EM的最新进展使复杂结构的建模成为可能I.结构研究提出了醌/抑制剂通路模型来解释醌/抑制剂如何与酶结合(Sazanov,2015;Wirth等,2016)。然而,由于目前还没有含有结合醌或抑制剂的复合物i的结构数据,因此通道模型是否反映了整个催化循环中存在的生理相关状态仍有争议。
光亲和标记技术是一种最常用的基于亲和性的蛋白质修饰方法(Hatanaka and Sadakane,2002),该技术使用具有光活性基团的合成配体,如二嗪和苯基叠氮。它为研究生物活性化合物与感兴趣蛋白质之间的相互作用提供了强有力的手段。最近,为了深入了解线粒体复合物i中醌/抑制剂结合位点的结构,我们使用光活性的[125i]阿米洛利([125i]pra3,[125i]pra4,[125i]pra5和[125i]pra6)与bovi进行了光亲和标记。NE心脏亚软骨颗粒(SMPS)(UNO等,2019年)。用放射性同位素(125i)作为示踪剂,使实验能以最低浓度的配体(1-10nm)进行。这些浓度比炔烃标记的胺碘化物(Pra1和Pra2,Murai等,2015年)低约两个数量级,交联后可通过铜催化的点击化学(Wang等,2003年)与生物素或荧光团结合。反应。这一优点可以最大限度地减少非特异性标记的可能性,这是假阳性结果的主要原因。
本文以[sup>125i]pra5为例(图1)描述了牛smps复合物i的光亲和标记的详细步骤,包括1)制备125i标记的光活性配体,2)用配体交联牛smps,3)鉴定标记的蛋白质。

关键字:光亲和标记, 呼吸链复合物I, 阿米洛利, 酶抑制剂, 线粒体, 生物能量学, NADH泛醌氧化还原酶, 化学生物学

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图1。用[125i]阿米洛利标记牛心脏smps的示意图

材料和试剂

  1. 1.5毫升螺旋盖微管(Watson,目录号:139-115-112C)
  2. 1.5毫升微管(安全锁管,Eppendorf,目录号:0030120086)
  3. 吸管头(Gilson)
  4. 膜过滤装置(Amicon Ultra,100 kDa,Merck Millipore,目录号:UFC510096)
  5. 反相高效液相色谱柱(Cosmosil 5C18-MSII,4.6 mm x 200 mm,Nacalai Tesque)
  6. 薄层色谱板(默克密理博,目录号:1.05554.0001)
  7. 保鲜膜
  8. 阿米洛利锡前体(A21a,1.0 mm乙醇,图1a,合成过程在UNO等中描述,2019年)
  9. [125i]nai(无载体,2000 ci/mmol,Perkin Elmer,目录号:NEZ033A)
  10. 氯胺T(Wako Pure Chemicals,目录号:032-02182)
  11. 亚硫酸氢钠(NaHSO3,Wako Pure Chemicals,目录号:198-01371)
  12. 牛亚软骨颗粒(SMPS)
    注:
    1. smps是线粒体内膜的内外囊泡。
    2. 线粒体是从牛心脏分离出来的,如别处所述(Smith,1967)。
    3. SMPS是在先前描述的方案(Matsuno Yagi和Hatefi,1985)的基础上,使用含有0.25 M蔗糖、1.0 m m琥珀酸钾、1.5 mm atp、10 mm mgcl2、10 mm mncl2和10 mm tris/hcl(pH7.4)的超声培养基制备的,然后进行超离心。在使用前,将其储存在含有250 mm蔗糖和10 mm三氯化三钠(pH7.4)的缓冲液中,温度为-80°C。
  13. TISP> TM双TIS蛋白凝胶(4-16%预制凝胶,THE Fisher Fisher,目录号:BN1004BOX)
  14. NativepageTM运行缓冲和阴极缓冲添加剂(Thermo Fisher,目录号:分别为BN2001和BN2002)
  15. Serva Blue G(CBB G-250,Serva,目录号:35050.01)
  16. n-十二烷基-d-β-麦芽糖苷(DDM,多津,目录号:D316)
  17. 甘油(Wako Pure Chemicals,目录号:075-00616)
  18. 乙醇(Wako Pure Chemicals,目录号:057-00456)
  19. 甲醇(Wako纯化学品,目录号:137-01823)
  20. 乙酸(Wako纯化学品,目录号:017-00251)
  21. 蔗糖(Wako Pure Chemicals,目录号:192-00017)
  22. mgcl2(六水,wako纯化学品,目录号:135-00165)
  23. NaCl(Wako纯化学品,目录号:195-01665)
  24. Ponceau S(Nacalai Tesque,目录号:28322-72)
  25. Tricine(Dojin,产品目录号:GB05)
  26. SDS(Wako Pure Chemicals,目录号:191-07145)
  27. 硫醇(Wako Pure Chemicals,目录号:137-06862)
  28. Tris(Wako Pure Chemicals,目录号:011-20095)
  29. 丙烯酰胺(Wako纯化学品,目录号:011-08015)
  30. 双丙烯酰胺(Wako纯化学品,目录号:134-15081)
  31. 尿素(Wako纯化学品,目录号:219-00175)
  32. 银染试剂盒(Wako Pure Chemicals,目录号:291-50301,与MS兼容)
  33. 三氟乙酸(TFA)
  34. NADH(东方酵母,产品目录号:4432万)
  35. 标签缓冲区(见配方)
  36. 4x BN页样本缓冲区(见配方)
  37. 洗脱缓冲液(见配方)
  38. 4x sch_gger样品缓冲液(见配方)
  39. 10倍阴极缓冲液(见配方)
  40. 10x阳极缓冲器(见配方)
  41. AB-3混合物(见配方)
  42. 3X凝胶缓冲液(见食谱)
  43. 1.0 m kpi缓冲液,ph值7.4(见配方)
  44. DSDS PAGE凝胶配方(见食谱)

设备

  1. 皮皮特曼(吉尔森)
  2. -80°C冷冻柜(PHC,型号:MDF-DU300H)
  3. 高效液相色谱系统(Shimazdu,型号:LC-10AS)
  4. 微型离心机(Chibitan,Merck Millipore,型号:XX42CF0RT)
  5. γ计数器(眼镜蛇TMII,帕卡德)
  6. 真空离心蒸发器(孔眼,型号:CVE-2200)
  7. 生物成像分析仪(分别为FLA-5100或台风FLA-9000、富士胶片或GE Healthcare)
  8. 成像板(BAS IP MS 2040E,GE Healthcare)
  9. 涡流混合器(一体式,型号:Trio TM-1N)
  10. 热块(一体式,型号:MYBL-10)
  11. 长波紫外线灯(UVP,黑色,型号B-100AP,配有100W灯泡,365纳米)
  12. 电洗脱器(Bio-Rad,型号:422)
  13. 凝胶干燥器(BIO RAD,型号5853,配备水工真空泵)
  14. 凝胶电泳仪和玻璃板(ATTO,AE-6500,8×9厘米,用于小型凝胶格式)

软件

  1. 图像分析软件,如Multi-Gauge或ImageQuant(分别为富士胶片或GE Healthcare)

程序

  1. 光活性[125i]阿米洛利的制备(以[125i]pra5为例)
    1. 将锡前体A21A(1.0 mm,20μl)的乙醇溶液制备在1.5 ml螺旋盖微管中。
    2. 添加[125i]nai(1 mci,2000 ci/mmol,10μl)。
      注:[125i]nai应在通风室中处理,直到反应被nahso3
    3. 通过添加水性氯胺T(3.0 m m,在1.0 m kpi缓冲液中,pH值7.4,10微升)开始反应。
      注:氯胺T水溶液应为每次实验新鲜制备。
    4. 旋涡并使管子向下旋转。
    5. 将混合物在室温下孵育10分钟。
    6. 加入5%(w/v)亚硫酸氢钠水溶液(10微升)使反应停止。
    7. 用手动进样口将混合物的整个体积(~50微升)注入高效液相色谱(循环体积应大于100微升)。信用证条件如下:
    8. 每30秒(约400微升)收集一次分数。
    9. 取3微升样品,分别用γ计数器(2微升)和放射薄层色谱(1微升)评估各组分的放射性和纯度(见图2)。
    10. 将含有[125i]pra5(frs)的分数合并。图2中的8和9),并通过真空离心蒸发器将样品浓缩至约50微升。
      注意:不要完全干燥样品。
    11. 用乙醇将[125i]pra5的浓度调整为1 mci/ml。
    12. 将样品储存在4°C([125i]阿米洛利作为乙醇溶液稳定至少2个月)。
      < BR>
      图2。反相高效液相色谱法纯化光活性[125i]pra5。将含有粗[sup>125i]pra5的混合物应用于c18柱。用10%甲醇/0.01%水性tfa(0-10分钟)冲洗柱去除盐,然后用90%甲醇/0.01%水性tfa(10-20分钟)等比例洗脱[125i]pra5。洗脱液每隔30s分馏一次,并将每个分馏液中的2微升小份转移到ria管中,使用γ计数器测量放射性(保留时间:10-17.5min)。a.高效液相色谱图中的放射性分布。放射化学产率从最初的[125i]nai约为70%。b.所收集组分的薄层色谱分析。以10%甲醇/氯仿为流动相,在薄层色谱板上分析样品(1微升)。在成像板上曝光1h,并用生物成像分析fla-5100进行分析。
      < BR>
  2. 牛脱脂乳粉的交联[sup>125i]pra5
    1. 在1.5毫升微管中,将牛SMPS悬浮液加入标记缓冲液(2.0-4.0毫克/毫升,100-200微升)。
    2. 添加浓度为5-10nm的[125i]pra5。
      注:乙醇浓度应低于~3%。
    3. 室温下孵育10分钟。
    4. 加入50μm NADH,然后孵育5分钟。
    5. 用长波紫外线灯在冰上照射10分钟,距离光源10厘米(图3)。
    6. 在最终浓度为1.0%的条件下,加入10%(w/v)ddm使交联反应猝灭。
    7. 将混合物在冰上孵育1h。
    8. 添加4x bn页样本缓冲区。
      < BR>
      图3。牛脱脂乳粉的光亲和标记装置。含脱脂乳粉的1.5毫升Eppendorf试管在冰上开盖放置。它们在离光源约10厘米的距离处紫外线照射10分钟。为了有效标记,样品的体积应为100-200微升/管。< BR> < BR>
  3. 用pra5标记的蛋白质的鉴定
    1. 蓝色本征(bn)page和电洗脱分离配合物i(sch_gger,2006)
      1. 添加5%Serva Blue G(CBB G-250)。CBB/DDM比率应按重量调整为1/4。
      2. 将样品加载到4-16%BN预制凝胶(100-200μg蛋白/阱)中。
      3. 开始电泳(100伏,15毫安,持续1小时)。
      4. 增加电压为200 V(15毫安),并继续电泳直到前面染料到达凝胶底部。
      5. 用剃刀刀片切除复杂的I型环。
        注意:不要固定BN凝胶。不包括任何未染色(或染色不良)凝胶。只切除复杂I波段的心脏。
      6. 根据制造商的协议组装电洗脱器。
      7. 用洗脱缓冲液填充玻璃管和洗脱液,将凝胶片放入管内(4-5凝胶片/管)。
      8. 在10毫安/玻璃管中洗脱蛋白质,在冷藏室中过夜。
        注:如果洗脱成功,凝胶片变为无色。
      9. 按照制造商的方案收集洗脱蛋白(我们建议用50-100微升0.5%十二烷基硫酸钠洗涤透析膜以溶解沉淀蛋白)。
      10. 用Amicon Ultra 0.5(100 kDa截止值)将收集的溶液浓缩至适当体积(50-100微升)。
      11. 将样品保存在-80°C直至使用。
    2. 用双SDS-PAGE分离复合I亚单位(DSDS-PAGE,RAIS等,2004;Wittig,2006)
      1. 将4x sch_gger样品缓冲液加入电洗脱分离的络合物i中。
      2. 将混合物在40℃下孵育1h。
        注意:不要煮沸样品,因为复合物I聚集了一些疏水亚基。
      3. 在电压为30 V(40 mA)的第一尺寸SCH-Guer-SDS凝胶(10% T,3% C,含有6 M尿素,4%层凝胶,表1)中分离复合的I蛋白。
      4. 当样品完全进入凝胶时,将电压增加至100 V,并继续电泳直到染料前端到达凝胶底部。
      5. 切除第一维凝胶条并在含有100毫米TrIS/HCl(pH 2)的酸性缓冲液中孵育30分钟,然后将条固定在两个玻璃板之间(参见图4A、4B和4C)。
      6. 倒入第二维度丙烯酰胺混合物(16%t,3%c,表1)并用水覆盖(见图4d)。
      7. 聚合后,向下推下第一层凝胶条,使两种凝胶粘在一起(见图4E)。
      8. 用丙烯酰胺混合物(16% T,3% C,表1)填充间隙,其中含有150毫米TrIS/HCl(pH 7.4)和痕量SeraBlue G(间隔胶,参见图4F)。
      9. 以30伏(40毫安)的电压开始第二次双向电泳。
      10. 当样品完全进入第二凝胶时,电压可以增加到150 V。
    3. 银染色和放射自显影
      1. 用合适的方法固定和染色银凝胶(严<EM>等<E/EM>2000)或市售银染试剂盒。
      2. 用传统平板扫描仪扫描银染凝胶。
      3. 将凝胶放置在凝胶干燥器之前,将凝胶在含有3%甘油、40%甲醇和10%乙酸的溶液中平衡30分钟。
      4. 用凝胶干燥器在65℃下干燥凝胶3小时。温度应缓慢升高[约1°C/min(“梯度模式”)]。
      5. 凝胶完全干燥后,用保鲜膜覆盖凝胶,然后将其暴露在成像板上12-24小时。
      6. 利用生物成像分析仪分析放射性标记蛋白的迁移模式,并与银染蛋白的迁移模式进行比较(典型结果如图5所示)。
        < BR>
        图4。DSDS PAGE的第二维凝胶的制备。< /强> A切除第一维凝胶条。小心地将凝胶条放入含有酸性缓冲液的15毫升猎鹰管中。在室温下将凝胶孵育30分钟。c.将玻璃条固定在两块玻璃板之间。将二级丙烯酰胺混合物倒入,在丙烯酰胺凝胶混合物聚合后,用MIL-LH<2</Sub>O. E.覆盖,将凝胶条推下,使两种凝胶粘在一起。f.移除MIL-LQ,然后用间隔胶填充间隙。< BR> < BR>
        图5。应用[sup>125i]pra5.a.复合物i的bn-page分离。在[416%BN凝胶]上分离[SUP>125</SUP> I] PRA5标记的SMPS。如别处所述(Muraiet al,2009;Shiraishiet al,2012),通过NADH/NBT系统的活性染色鉴定复合I带,并进行电洗脱。大约100微克SMPS蛋白质被装载到每个孔中。b.通过DSD页解析复杂I。用BN-PAGE和电洗脱纯化的[SUP> 125 I] PRA5标记的复合物I,在10%维SCH型GuGER型三萜凝胶(10% T,3% C含有6 M脲)上分离,随后在16% SaωGGER型凝胶(16% T,3% C)上进行第二次分离。2D凝胶经银染(左侧)和放射自显影(右)。[125i]pra5的标记提供了两个与49kda和psst亚单位相对应的放射性点,这两个亚单位都包含复合物i的醌/抑制剂结合囊(hirst 2013和sazanov 2015)。这些斑点通过质谱和western blotting鉴定(Shiraishi等,2012)。我们注意到,ADP/ATP载体(AAC)的蛋白斑点中有微弱的放射性,这是用络合物I纯化的。I的凝胶图像与参考图7C中所用的凝胶图像相同(UNO<EM>等</EM>2019)。

食谱

  1. 标记缓冲液(用于牛脱脂奶粉的紫外线照射)
    250毫米蔗糖
    50毫米关键绩效指标(pH值7.4)
    1毫米mgcl2
  2. 4x bn页样本缓冲区
    200 mm双Tris/HCl(pH 7.2)
    200毫米氯化钠
    40%(w/v)甘油
    0.004%(w/v)胭脂红
  3. 洗脱缓冲液(用于络合物i的电洗脱)
    25毫米三角线
    7.5 mm双Tris/HCl(在4°C下调整pH值7.0)
  4. 4x sch_gger样品缓冲液(用于tricine页)
    150毫米Tris/HCl(pH 7.0)
    12%(w/v)十二烷基硫酸钠
    30%(w/v)甘油
    6%(w/v)巯基乙醇
    0.05%(w/v)Serva蓝色G
  5. 10x阴极缓冲液(用于Sch_gger型SDS-PAGE)
    1.0米Tris
    1.0米Tricine
    1% SDS
  6. 10x阳极缓冲液(用于Sch_gger型SDS-PAGE)
    1.0 m Tris/HCl(pH 8.9)
  7. AB-3混合物(49.5%T,3%C,用于Sch_gger型SDS-PAGE)
    48%丙烯酰胺
    1.5%双丙烯酰胺
  8. 3X凝胶缓冲液(用于SCH-Guer-SDS-PAGE)
    3.0 m Tris/HCl(pH 8.45)
    0.3%十二烷基硫酸钠
  9. 1.0 m kpi缓冲液,ph值7.4
    将k2hpo4(12.1 g,69.5 mmol)和kh2po4(4.1 g,30.4 mmol)溶于100 ml蒸馏水中。
  10. DSDS PAGE凝胶组合物(SHC-Guger-SDS-PAGE)(表1)
    < BR> 表1。sch_gger型sds凝胶的制备(用于两种阿托微凝胶)

    < BR>

致谢

确认本协议改编自Uno等。(2019年)。这项研究得到了JSPS Kakenhi的支持(批准号JP26292060和JP18H02147给H.M.,批准号JP18K05458给M.M.)。涉及放射性同位素技术的实验在京都大学放射性同位素研究中心进行。

相互竞争的利益

作者声明他们与本文内容没有利益冲突。

工具书类

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Copyright: © 2019 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. Murai, M. and Miyoshi, H. (2019). Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [125I] amilorides. Bio-protocol 9(17): e3349. DOI: 10.21769/BioProtoc.3349.
  2. Uno, S., Kimura, H., Murai, M. and Miyoshi, H. (2019). Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches. J Biol Chem 294(2): 679-696.
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