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Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803

分析集胞藻属PCC6803全细胞、类囊体和光合蛋白复合体中的金属

CG Chiara Gandini
Søren Husted Søren Husted
SS Sidsel Birkelund Schmidt

发布: 2018年06月20日第8卷第12期 DOI: 10.21769/BioProtoc.2889 浏览次数: 6574

评审: Nidhi SharmaAnonymous reviewer(s)

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Abstract

Metals are essential in many biological processes, including oxygenic photosynthesis. Here we described a method to measure the metal pool in whole cells and thylakoids, including the bioactive pool in intact photosynthetic protein complexes in the model oxygenic cyanobacterium Synechocystis PCC6803. In the first part of the protocol, whole cells and thylakoid membranes are carefully prepared, in which the total metal concentrations are measured by inductively coupled plasma triple-quadrupole mass spectrometry (ICP-QQQ-MS). In the second part of the protocol, isolated thylakoids are solubilized to release the integral membrane proteins and the metal binding protein complexes. These intact photosynthetic protein complexes are subjected to size exclusion chromatography (SEC) and metal binding in the size separated complexes is analyzed by hyphenation with ICP-QQQ-MS.

Keywords: Cyanobacteria (蓝藻) search Synechocystis (集胞藻) search Manganese (锰) search Iron (铁) search Magnesium (镁) search Thylakoid membranes (类囊体膜) search Speciation analysis (形态分析) search ICP-MS (ICP-MS) search

Background

The process of oxygenic photosynthesis requires metals due to their essential functions as cofactors and catalysts in the photosynthetic electron transport chain. The photosynthetic apparatus requires iron (Fe) in the form of either Fe-S clusters, heme-bridged Fe and non-heme Fe, copper (Cu) in the soluble mobile electron carrier protein plastocyanin, magnesium (Mg) in chlorophylls, calcium (Ca) and manganese (Mn) in the oxygen evolving complex of photosystem II (PSII). Tight control of metal allocation inside photosynthetic cells is essential for cell survival since an imbalanced metal accumulation induces mismetallation and inactivation of the various metallo-enzymes. An accurate analysis of the concentration and allocation of metals in photosynthetic cells is therefore important to investigate the role of key factors and proteins involved in metal homeostasis. While the method described in Brandenburg et al. (2017) accurately quantifies the periplasmic and intracellular pools of Mn in Synechocystis cells, the protocol presented here provides a wider overview, quantifying metals in whole Synechocystis cells, isolated thylakoids, and the bioactive metal pool in fractionated photosynthetic complexes. This protocol is partially based on the method described in Schmidt et al. (2015) for barley thylakoids.

Materials and Reagents

  1. Pipette tips
  2. 50 ml conical tubes (Greiner Bio One International, catalog number: 227261 )
  3. 1.5 and 2 ml Eppendorf tubes (Eppendorf, catalog numbers: 0030120086 and 0030120094 , respectively)
  4. Microwave Teflon tubes, 8 ml (VWR, catalog number: 525-0178 )
  5. Nylon membrane filters, 0.45 µm (Frisenette, catalog number: 13NY045-100 )
  6. Syringe, 1 ml (Frisenette, catalog number: 9161406 )
  7. NanoVipers fingertight fittings, 150 µm (Thermo Fisher Scientific, catalog number: 6041.5820 )
  8. Synechocystis sp. PCC6803 GT (glucose tolerant strain, from Dr. Himadri Pakrasi laboratory, Department of Biology, Washington University, St Louis, MO, USA)
  9. Multi-metal calibration standards for ICP-MS calibration (CPI International, catalog numbers: P/N4400-132565A and P/N4400-132565B )
  10. EDTA, BioUltra (Sigma-Aldrich, catalog number: E1644-100G )
  11. Tricine (Sigma-Aldrich, catalog number: T9784-25G )
  12. Lysozyme (Sigma-Aldrich, catalog number: 62971-50G-F )
  13. Sucrose, BioExtra (Sigma-Aldrich, catalog number: S7903-1KG )
  14. Sodium chloride (NaCl), BioExtra (Sigma-Aldrich, catalog number: S7653-250G )
  15. Magnesium chloride (MgCl2·7H2O), BioExtra (Sigma-Aldrich, catalog number: M2670-1KG )
  16. Na-ascorbate, BioExtra (Sigma-Aldrich, catalog number: 11140-250G )
  17. Sodium fluoride (NaF), BioExtra (Sigma-Aldrich, catalog number: S7920-100G )
  18. Liquid nitrogen
  19. PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, catalog number: 23225 )
  20. Glass beads (Sigma-Aldrich, catalog number: G4649 )
  21. Milli-Q water
  22. PlasmaPURE 67-69% HNO3 (SCP SCIENCE, catalog number: 250-039-175 )
  23. 30% H2O2 (prepare 15% H2O2 with Milli-Q water) (Sigma-Aldrich, catalog number: 31642 )
  24. n-Dodecyl-β-D-Maltopyranoside (β-DM) (Anatrace, catalog number: D310LA )
  25. Bis-Tris, BioExtra (Sigma-Aldrich, catalog number: B7535-100G )
  26. Glycerol, BioExtra (Sigma-Aldrich, catalog number: G6279 )
  27. Betaine, BioUltra (Sigma-Aldrich, catalog number: 61962-250G )
  28. Pefabloc SC (Roche Diagnostics, catalog number: 11429868001 )
  29. Apple leaves, standard reference material (Sigma-Aldrich, catalog number: NIST1515 )
  30. Glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  31. Na2S2O3·5H2O (Merck, catalog number: 1.06516.0500 )
  32. FeNH4 citrate (MP Biomedicals, catalog number: 02158040 )
  33. Na2CO3 (Sigma-Aldrich, catalog number: S7795-500G )
  34. K2HPO4 (Sigma-Aldrich, catalog number: P8281 )
  35. NaNO3 (Sigma-Aldrich, catalog number: S8170-250 )
  36. MgSO4 (Sigma-Aldrich, catalog number: M2643-500G )
  37. CaCl2 (Sigma-Aldrich, catalog number: C5670-500G )
  38. Citric acid (Sigma-Aldrich, catalog number: 251275-100G )
  39. Na2-EDTA (Sigma-Aldrich, catalog number: E4884-500G )
  40. H3BO3 (Sigma-Aldrich, catalog number: B6768-500G )
  41. MnCl2 (Sigma-Aldrich, catalog number: 244589-50G )
  42. ZnSO4 (Sigma-Aldrich, catalog number: Z0251-100G )
  43. NaMoO4 (Sigma-Aldrich, catalog number: M1003-100G )
  44. CuSO4 (Sigma-Aldrich, catalog number: 209198-100G )
  45. Co(NO3)2 (Alfa Aesar, catalog number: 36418.22 )
  46. BG11-G (see Recipes)
  47. 100x BG-FPC (see Recipes)
  48. 1,000x trace element stock (see Recipes)
  49. FeNH4 citrate stock (see Recipes)
  50. Na2CO3 stock (see Recipes)
  51. K2HPO4 stock (see Recipes)
  52. EDTA washing buffer (see Recipes)
  53. Tricine buffer (see Recipes)
  54. Lysozyme solution (see Recipes)
  55. Homogenization buffer (see Recipes)
  56. Tricine-NaF buffer (see Recipes)
  57. Storage buffer (see Recipes)
  58. Solubilization buffer (see Recipes)
  59. Detergent solution (see Recipes)
  60. Mobile phase buffer (see Recipes)

Equipment

  1. Pipettes
  2. Erlenmeyer flasks, 2 L (Fisher Scientific, catalog number: 11961566)
    Manufacturer: Pyrex, catalog number: 1130/30D .
  3. Orbital shaker (Eppendorf, New BrunswickTM, model: Innova® 2150, catalog number: M1194-0010 )
  4. Eppendorf centrifuge 5418 R, refrigerated (Eppendorf, model: 5418 R, catalog number: 5401000013 )
  5. Eppendorf centrifuge 5430 R, refrigerated (Eppendorf, model: 5430 R, catalog number 5428000210 )
  6. Beckman Coulter centrifuge (Beckman Coulter, model: Avanti® J-25 ) with type 19 rotor, fixed angle
  7. 250 ml bottles (Beckman Coulter, catalog number: 325620 )
  8. Microfluidizer Processor (Microfluidics, model: M-110L )
  9. Light microscope (ZEISS, model: Axiovert 135 TV )
  10. Bead mill TissueLyser II (QIAGEN, catalog number: 85300 )
  11. TissueLyser II adapter set (QIAGEN, catalog number: 69982 )
  12. Heraeus Fresco 21 Microcentrifuge, refrigerated (Thermo Fisher Scientific, catalog number: 75002425 )
  13. HPLC Microvials PP, 300 µl and screw cap PP, PFTE (VWR, catalog numbers: 548-0440 and 548-0787 )
  14. Size exclusion column, Biobasic SEC-1000 analytical column (300 x 7.8 mm) and guard column (30 x 7.8 mm), 5 µm particle size, 1000Å pore size (Thermo Fisher Scientific, catalog number: 73605-307846 and 73605-037821 )
  15. Pressurized Ultrawave System (Ultrawave system, Milestone Srl, Sorisole, Italy)
  16. HPLC system, bio-inert (i.e., the mobile phase and samples have no contact with metal parts) (Thermo Fisher Scientific, Dionex, model: UltiMate 3000 )
  17. DionexTM ICS-5000+ DP Dual Pumps (Thermo Fisher Scientific, model: ICS-5000+ DP )
  18. ICP-QQQ-MS (Agilent Technologies, model: 8800 Triple Quadrupole ICP-MS )
  19. Nebulizer, Ari Mist HP (Burgener Research, catalog number: AM HP 5500 )
  20. Gases for ICP: Liquid Argon, grade 5.0, and reaction gas: 20% Oxygen in Argon (AGA, catalog numbers: 107407 and 714039 )

Software

  1. MassHunter 4.2 Workstation software 8800 ICP-QQQ data analysis (Agilent Technologies)

Procedure

English
中文翻译

文章信息

版权信息

© 2018 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Gandini, C., Husted, S. and Schmidt, S. B. (2018). Analysis of Metals in Whole Cells, Thylakoids and Photosynthetic Protein Complexes in Synechocystis sp. PCC6803. Bio-protocol 8(12): e2889. DOI: 10.21769/BioProtoc.2889.

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分类

植物科学 > 植物生物化学 > 其它化合物

植物科学 > 植物生理学 > 光合作用

生物化学 > 其它化合物 > 元素

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