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Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria
   

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本实验方案简略版
Molecular Microbiology
Feb 2015

Abstract

The topology of membrane proteins and enzymes can be determined using various methods including reporter protein fusions and accessibility of cysteine residues to alkylating agents. Here we describe a variation of the substituted cysteine accessibility method to determine membrane topology and activity of enzymes containing an active site cysteine. Membrane topology of proteins can be predicted using different programs and the actual membrane topology can be determined by monitoring the accessibility of cysteine residues introduced in periplasmic (exposed) or cytoplasmic (not exposed) loops to alkylating agents. A two-step protocol is described where whole Escherichia coli (E. coli) cells are first treated with or without a membrane impermeable thiol reagent (2-sulfonatoethyl)-methane thiosulfonate (MTSES) and subsequently labeled with an alkylating reagent maleimide polyethyleneglycol (malPEG). When cysteine residues are accessible to MTSES, and thus exposed to (or accessible from) the periplasm, their free thiol groups covalently react with MTSES and consequently, are blocked for alkylation with malPEG. The thiol groups of cytoplasmic or membrane-embedded cysteine residues are not accessible to MTSES and proteins can be alkylated with malPEG resulting in an increase in molecular weight of 5 kDa. In the second part of the protocol, accessibility of cysteine residues is used to address the acylation state of enzymes that form stable thioester acyl intermediates. Thioesters can be specifically cleaved by neutral hydroxylamine, leading to a free thiol group of the active site cysteine that can then be alkylated with malPEG.

Materials and Reagents

  1. Escherichia coli K12 cells
  2. Distilled sterile water (dH2O)
  3. Acetone (Sigma-Aldrich, catalog number: 24201-1L-R )
  4. Chloroform (CHCl3) (VWR International, catalog number: 22711.290 )
  5. Trichloroacetic acid (TCA) (Sigma-Aldrich, catalog number: 27242-500G-R )
  6. Ethanol (EtOH) (100%) (Sigma-Aldrich, catalog number: 24103-5L-R )
    Note: Product 24103 has been discontinued.
  7. Tris(hydroxymethyl)aminomethane hydrochloride (Trizma base) (Sigma-Aldrich, catalog number: 337411KG )
  8. 1 M KH2PO4 (Merck Millipore Corporation, catalog number: A148973 )
  9. 1 M Potassium phosphate dibasic K2HPO4 (Fluka, catalog number: 60353 )
    Note: Currently, it is “ Sigma-Aldrich, catalog number: 60353 ”.
  10. Sodium dodecyl sulfate 20%SDS 20% (biosolve-chemicals, catalog number: 19812323 )
  11. Urea (Merck Millipore Corporation, Calbiochem®, catalog number: 66612 )
  12. NaOH (Merck Millipore Corporation, catalog number: 21778425 )
  13. Bromophenol blue (Sigma-Aldrich, catalog number: 114391 )
  14. Glycerol (VWR International, catalog number: 24388.295 )
  15. Parafilm (Thomas Scientific, Pichiney Plastic Packaging, catalog number: PM-992 )
  16. 50 mM Phosphate buffer (PB) (pH 7.0) (see Recipes)
  17. 250 mM Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich,catalog number: ED2SS ) (see Recipes)
  18. 0.3 M Sodium (2-sulfonatoethyl) methane thiosulfonate (MTSES) (Anatrace, catalog number: S110MT ) (see Recipes)
  19. 1 M L-cysteine (IGN, catalog number: 194646 ) (see Recipes)
    Note: Currently, it is “mpbio, catalog number: 194646 ”.
  20. Methanol (MeOH) (95%) (Sigma-Aldrich, catalog number: 32213-1L ) (see Recipes)
  21. 1 M Tris-HCl (see Recipes)
  22. Denaturation buffer (see Recipes)
  23. PEG buffer (see Recipes)
  24. 1.2 mM Methoxypolyethylene glycol 5000 maleimide (malPEG) (Sigma-Aldrich, catalog number: 63187 ) (see Recipes)
  25. Hydroxylamine solution (HA) (Sigma-Aldrich, catalog number: 59417 ) (see Recipes)
  26. 1 M Tris.HCl, 1% SDS (see Recipes)
  27. SDS-Sample buffer (see Recipes)
  28. DL-Dithiothreitol (DTT) (Sigma-Aldrich, catalog number: D9779 ) (see Recipes)

Equipment

  1. Incubator (37 °C) (Infors AG)
  2. Spectrophotometer (600 nm) (Eppendorf, model: Biophotometer plus )
  3. Tabletop centrifuge (Eppendorf, model: 5472R/5427R )
  4. Gelectrophoresis apparatus (Bio-Rad Laboratories, model: Miniprotean II )
  5. Western blot apparatus (Bio-Rad Laboratories, model: Transblot Turbo )
  6.  Vortex Genie 2 (Scientific Industries)

Procedure

I. Accessibility of cysteine residues using thiol specific and alkylating reagents

  1. Culture and induction
    The culture conditions depend on the expression system and bacterial species used. For E. coli, IPTG inducible promoter such as pLac or L-arabinose inducible pBAD and systems alike are highly recommended. Use a single colony from a fresh plate streaked from -80 °C stock to inoculate a small volume (5 ml) for an overnight culture. Induce expression of the gene of interest in early exponential phase (OD600 around 0.2 in LB medium) and let bacteria grow at 37 °C till late exponential (2 h, OD600 around 0.6-0.7) to produce recombinant protein. This method has to be determined for the protein of interest. Measure absorbance at 600 nm (A600) at the end of induction period and chill culture on slushy ice. Slushy ice is 4 °C and cools the cell culture down quickly. The protocol is based on 1 ml of cells with an A600 of 1.0 (approximately 5 x 108-1 x 109 cells).

  2. Preparation of samples for MTSES protection
    Prior to experiment: Remove MTSES from -20 °C and let it warm up to room temperature, prepare fresh denaturation buffer and PEG buffer. All steps are performed at room temperature unless stated otherwise.
    1. Centrifuge 2 x 1 ml of cell culture for 1 min. at 16,100 x g in tabletop centrifuge. Wash cells in 1 ml PB.
    2. Centrifuge cells. Resuspend cell pellet in 0.24 ml PB buffer and add 10 μl 25 mM EDTA (1 mM final). Use cells directly in step C.
      Note: EDTA treatment permeabilizes the outer membrane.
    3. For each sample, aliquot 120 μl into microcentrifuge tubes labeled with (+) and without (-) MTSES.

  3. MTSES protection
    Note: Treat samples as gently as possible while exposed to MTSES. EDTA permeabilizes the outer membrane, but the cytoplasmic membrane needs to be intact. After addition of L-cysteine to quench the reaction, samples can be treated more roughly. Wash steps are to remove L-cysteine.
    1. Add 13 μl 0.3 M MTSES (30 mM final) to tube labeled (+) MTSES.
    2. Add 13 μl dH2O to tube labeled (-) MTSES by carefully pipetting up and down.
    3. Incubate 30 min at room temperature.
    4. Add 14 μl 1 M L-cysteine (100 mM final) to both tubes by carefully pipetting up and down.
    5. Incubate 10 min at room temperature.
    6. Add 0.75 ml PB.
    7. Centrifuge for 5 min at 16,100 x g in tabletop centrifuge.
    8. Wash the cells 2x with 0.5 ml PB.
    9. Resuspend cells in 50 μl PB.

  4. Sample denaturation with methanol/chloroform
    Note: Pre-cool tabletop centrifuge to 4 °C.
    1. Add 750 μl of Denaturation Buffer.
    2. Vortex vigorously, incubate at 4 °C for 30 min.
    3. Spin at 16,100 x g for 3 min.
    4.  Remove supernatant very carefully and completely with pipette. An additional centrifugation step is usually not required.
    5.  Add 400 μl 95% MeOH.
    6. Spin at 16,100 x g for 4 min.
    7. Remove supernatant carefully and air-dry pellets for 15 min at room temperature.
      Note: Do not invert tubes, pellets are loose.

  5. Alkylation with malPEG
    Note: Bring bottle with malPEG to room temperature and make malPEG solution, protect from light. Put malPEG powder back at -20 °C after wrapping bottle with Parafilm.
    1. Cool 100% EtOH on ice.
    2. Resuspend each air-dried pellet in 100 μl PEG buffer, vortex.
    3. Add 20 μl malPEG to each tube and mix carefully by pipetting up and down.
    4. Incubate at room temperature for 30 min in the dark.
    5. Add 1.5 ml ice cold 100% EtOH and freeze at -20 °C for 1 h.
      Note: Samples can be left at -20 °C overnight.

  6. SDS-PAGE and Western blotting
    Note: Before starting, pre-cool tabletop centrifuge.
    1. Pellet samples for 15 min at 16,100 x g in tabletop centrifuge.
    2. Carefully remove most of the supernatant.
    3. Re-centrifuge samples and remove all left over liquid.
    4. Air dry pellets.
      Note: Do not invert tubes, pellets are loose.
    5. Resuspend samples in SDS-PAGE sample buffer containing 4 mM DTT.
    6. Heat at 100 °C for 5 min.
    7. Load equivalent amounts on gel using A600 measurements at time of sample preparation.
    8. Load each -/+ pair adjacent to each other.
    9. Run gel and Western-blot.

II. Accessibility of cysteine to alkylating agents after cleavage of thioester bond

Follow protocol described in A.

  1. HA treatment
    1. Centrifuge 1.6 ml of cell culture for 1 min at 16,100 x g in tabletop centrifuge.
    2. Resuspend cells in 1 ml PB.
    3. Divide in 2 x 500 μl in tubes labeled – and + HA and centrifuge cells.
    4. Resuspend cells in 500 μl 1M Tris-HCl (pH 7.0), 1% SDS (-) or 500 μl 1 M HA (pH 7.4), 1% SDS (+) by pipetting up and down.
      Note: This denatures proteins and cleaves thioester bonds in one step.
    5. Incubate at room temperature for 30 min.
    6. Add 50 μl 100% TCA, vortex.
    7. Incubate on ice for 30 min. Pre-cool tabletop centrifuge.
    8. Centrifuge protein precipitate for 15 min at 16,100 x g at 4 °C.
    9. Wash pellet 3 x with 1 ml ice cold acetone (stored at -20 °C) and centrifuge 10 min at 16,100 x g each time.
    10. Air dry pellets. Follow steps in E and F.
      Note: TCA pellets are often difficult to dissolve. Vortex rigorously and frequently.

Representative data


Figure 1. Example of malPEG alkylation profile of Apolipoprotein N-acyltransferase (Lnt) of E. coli.
Cysteine residues 23 and 62 were substituted by alanine, leaving a single active site cysteine residue C387. Lnt forms a thioester acyl-enzyme intermediate in vivo with C387 and is blocked for reaction with malPEG. Upon treatment of cells with hydroxylamine (HA), the thioester bond is cleaved, leaving a free thiol group of C387 and alkylation by malPEG. Please see Gélis-Jeanvoine et al. (2015) for representative examples.

Notes

Results may vary depending on the membrane protein studied. It is highly recommended that negative and positive controls are included to validate technical details of the procedure. For example, use of empty vector, a cysteine-less variant of the protein of interest and inactive mutants with substituted cysteine residues are recommended.

Recipes

  1. Phosphate buffer (PB) (50 mM, pH 7.0)
    Make 100 ml 1 M KH2PO4
    Make 100 ml 1 M K2HPO4
    Mix 1.5375 ml K2HPO4 with 0.9625 ml KH2PO4 and add distilled sterile water to 50 ml
    The final concentration is 50 mM KPi (pH 7.0)
  2. 0.25 M EDTA
    Add 93.05 g EDTA to 800 ml of H2O
    Adjust pH to 8.0 with NaOH
    Note: Disodium EDTA will not go in solution until the pH around 8.0.
    Divide into aliquots and autoclave
  3. MTSES solution (make fresh before use)
    For each 36.3 mg add 500 μl H2O, final concentration is 0.3 M
  4. 1 M L-cysteine
    121 mg L-cysteine in 1 ml H2O
  5. 95% MeOH
    To 47.5 ml MeOH add H2O to 50 ml
  6. 1 M Tris.HCl
    Add 121.1 g Trisma base into 800 ml H2O, adjust to pH 7.0 with HCl
    Add H2O to final volume of 1 L
    Autoclave
  7. Denaturation buffer (make fresh before use)
    To obtain a solution of H2O:MeOH:CHCl3 = 1:4:1, add to a 15 ml Falcon tube:
    2 ml H2O
    8 ml MeOH
    2 ml CHCl3
  8. PEG buffer
    Contains 10 M urea, 1% SDS, 1 mM EDTA in 1 M Tris pH 7.0.
    Per ml solution:
    0.6 g urea
    0.01 g SDS
    Dissolve in 600 μl 1 M Tris (pH 7.0), requires mixing and immersion in warm water
    Do not put the solution on ice after preparation because urea will precipitate
    Add 4 μl 0.25 M EDTA
    Adjust volume to 1 ml with 1 M Tris (pH 7.0)
    Note: PEG buffer contains urea, SDS and EDTA that denatures all proteins and renders all unprotected cysteine residues accessible to malPEG.
  9. malPEG solution
    Note: Make fresh before use, can not be stored.
    Allow malPEG to get to room temperature
    Weigh a small quantity, around 5 mg, on weigh paper
    Add H2O to reach a final concentration of 1.2 mM MalPEG: Add 170 μl of H2O for 1 mg of malPEG
    Note: For each sample 10 μl of 1.2 mM malPEG is needed, which corresponds to 12 nmol malPEG. 1 nmol malPEG is 5 μg. The minimum amount of malPEG to weigh accurately is 4-5 mg. This is enough for 34 samples.
  10. Hydroxylamine solution (HA)
    Dissolve 1.4 g HA in 10 ml of cold H2O to make 20 ml final of HA solution
    Adjust the pH to 7.4 with 32% NaOH as follows
    Add 1.5 ml of 32% NaOH, stir and check pH
    Gradually add more NaOH and check pH till a pH of 7.4 is reached
    Bring volume to 20 ml with H2O
    Note: The pH should be between 6.9 and 7.5, around pH 8.5 solution loses specificity.
  11. 1 M Tris.HCl, 1% SDS
    Add 20% SDS to 1 M Tris solution to reach a final concentration of 1%
  12. SDS-Sample buffer (4x)
    2.5 ml 1 M Tris (pH 6.8)
    1.0 g SDS
    4 ml 100% glycerol
    0.8 ml 0.1% bromophenol blue
    0.16 ml 1 M DTT
    Adjust total volume to 10 ml with dH2O
  13. 1 M DTT
    Dissolve 1.5 g DTT in 8 ml H2O, adjust volume to 10 ml
    Divide in 1 ml aliquots and store at -20 °C

Acknowledgments

This work was financed by the Institut Pasteur.

References

  1. Bogdanov, M., Zhang, W., Xie, J. and Dowhan, W. (2005). Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis. Methods 36(2): 148-171.
  2. Gélis-Jeanvoine, S., Lory, S., Oberto, J. and Buddelmeijer, N. (2015). Residues located on membrane-embedded flexible loops are essential for the second step of the apolipoprotein N-acyltransferase reaction. Mol Microbiol 95(4): 692-705.

简介

膜蛋白和酶的拓扑学可以使用各种方法确定,包括报告蛋白融合和半胱氨酸残基对烷化剂的可达性。在这里,我们描述了取代的半胱氨酸可接近性方法的变化,以确定膜拓扑和含有活性位点半胱氨酸的酶的活性。可以使用不同的程序预测蛋白质的膜拓扑,并且可以通过监测在周质(暴露的)或细胞质(未暴露的)环中引入的半胱氨酸残基对烷化剂的可及性来确定实际的膜拓扑。描述了两步方案,其中首先用或不用膜不可渗透的硫醇试剂(2-磺酸基乙基) - 甲烷硫代磺酸盐处理整个大肠杆菌(大肠杆菌)细胞(MTSES)并随后用烷基化试剂马来酰亚胺聚乙二醇(malPEG)标记。当半胱氨酸残基可接近MTSES并且因此暴露于周质(或可从周质接近)时,它们的游离硫醇基团与MTSES共价反应,并因此被malPEG封闭以进行烷基化。胞质或膜嵌入的半胱氨酸残基的硫醇基团不能到达MTSES,并且蛋白质可以用malPEG烷基化,导致5kDa的分子量增加。在方案的第二部分中,半胱氨酸残基的可及性用于解决形成稳定的硫酯酰基中间体的酶的酰化状态。硫酯可以被中性羟胺特异性切割,导致活性位点半胱氨酸的游离巯基,然后可以用malPEG烷基化。

材料和试剂

  1. 大肠杆菌 K12细胞
  2. 蒸馏无菌水(dH 2 O)
  3. 丙酮(Sigma-Aldrich,目录号:24201-1L-R)
  4. <氯仿(CHCl 3)(VWR International,目录号:22711.290)
  5.  三氯乙酸(TCA)(Sigma-Aldrich,目录号:27242-500G-R)
  6. 乙醇(EtOH)(100%)(Sigma-Aldrich,目录号:24103-5L-R) 注意:产品24103已停用。
  7. 三(羟甲基)氨基甲烷盐酸盐(Trizma碱)(Sigma-Aldrich,目录号:337411KG)
  8. 1 M KH 2 PO 4(Merck Millipore Corporation,目录号:A148973)
  9. < 1M磷酸氢二钾K 2 HPO 4(Fluka,目录号:60353)
    注意:目前,它是"Sigma-Aldrich,目录号:60353"。
  10. 十二烷基硫酸钠20%SDS 20%(生物溶剂化学品,目录号:19812323)
  11.  尿素(Merck Millipore Corporation,Calbiochem ,目录号:66612)
  12. < NaOH(Merck Millipore Corporation,目录号:21778425)
  13.  溴酚蓝(Sigma-Aldrich,目录号:114391)
  14. 甘油(VWR International,目录号:24388.295)
  15.   Parafilm(Thomas Scientific,Pichiney Plastic Packaging,目录号:PM-992)
  16.   50 mM磷酸盐缓冲液(PB)(pH 7.0)(请参阅配方)
  17. 250mM乙二胺四乙酸(EDTA)(Sigma-Aldrich,目录号:ED2SS)(参见配方)
  18.   0.3 M(2-磺酸基乙基)甲烷硫代磺酸钠(MTSES)(Anatrace,目录号:S110MT)(参见配方)
  19.   1 M L-半胱氨酸(IGN,目录号:194646)(请参阅配方)
    注意:目前,它是"mpbio,目录号:194646"。
  20.  甲醇(MeOH)(95%)(Sigma-Aldrich,目录号:32213-1L)(参见配方)
  21.   1 M Tris-HCl(请参阅配方)
  22.  变性缓冲区(请参阅配方)
  23. PEG缓冲液(见配方)
  24. > 1.2mM甲氧基聚乙二醇5000马来酰亚胺(malPEG)(Sigma-Aldrich,目录号:63187)(参见配方)
  25.  羟胺溶液(HA)(Sigma-Aldrich,目录号:59417)(参见配方)
  26. 1M Tris.HCl,1%SDS(参见配方)
  27. SDS-样品缓冲液(参见配方)
  28. DL二硫苏糖醇(DTT)(Sigma-Aldrich,目录号:D9779)(参见配方)

设备

  1. 孵育器(37℃)(Infors AG)
  2. 分光光度计(600nm)(Eppendorf,型号:Biophotometer plus)
  3. 台式离心机(Eppendorf,型号:5472R/5427R)
  4. 电泳装置(Bio-Rad Laboratories,型号:Miniprotean II)
  5. Western印迹装置(Bio-Rad Laboratories,型号:Transblot Turbo)
  6.   Vortex Genie 2(科学工业)

程序

我。使用硫醇特异性和烷基化试剂的半胱氨酸残基的可及性

  1. 离心2×1ml细胞培养物1分钟。在16,100×g 在台式离心机。洗涤细胞在1ml PB。
  2. 离心细胞。重悬细胞沉淀在0.24毫升PB缓冲液中,加入 ?10μl25mM EDTA(1mM终浓度)。在步骤C中直接使用单元格。
    注意:EDTA处理渗透外膜。
  3. 对于每个样品,等分120μl到用(+)和没有( - )MTSES标记的微量离心管中。

  • MTSES保护
    注意:在暴露于MTSES时,尽可能轻柔地处理样品。 EDTA 透化外膜,但细胞质膜需要 完好无损。加入L-半胱氨酸淬灭反应后,样品 可以更粗略地对待。清洗步骤是除去L-半胱氨酸。
    1. 加入13μl0.3 M MTSES(最终30 mM)到管标记(+)MTSES
    2. 小心地向上和向下移液,向试管标记的( - )MTSES中加入13μldH 2 O。
    3. 在室温下孵育30分钟。
    4. 通过小心地向上和向下移液,向两个管中加入14μl1μML-半胱氨酸(终浓度100mM)
    5. 在室温下孵育10分钟。
    6. 加入0.75ml PB。
    7. 在台式离心机中以16,100×g离心5分钟。
    8. 用0.5 ml PB洗涤细胞2次。
    9. 重悬细胞在50微升PB。

  • 用甲醇/氯仿进行样品变性
    注意:预先冷却的台式离心机至4°C。
    1. 加入750μl变性缓冲液。
    2. 剧烈涡旋,在4℃孵育30分钟
    3. 旋转16,100 x g 3分钟。
    4.  用移液管去除上清液非常仔细和完全。通常不需要另外的离心步骤。
    5.  加入400μl95%MeOH
    6. 旋转16,100 x g 4分钟。
    7. 小心地除去上清液,并在室温下风干颗粒15分钟 注意:不要翻转管,球团松动。

  • 用malPEG烷基化
    注意:将带有malPEG的瓶子带到室温并制备malPEG 溶液,防光。将malPEG粉末放回-20°C后 包装有石蜡膜的瓶子。
    1. 在冰上冷却100%EtOH
    2. 将每个空气干燥的沉淀重悬于100μlPEG缓冲液中,涡旋
    3. 每管加入20μlmalPEG,并通过上下吹打小心混合
    4. 在室温下在暗处孵育30分钟。
    5. 加入1.5ml冰冷的100%EtOH,在-20℃下冷冻1小时 注意:样品可以在-20°C过夜。

  • SDS-PAGE和Western印迹 注意:开始之前,预先冷却台式离心机。
    1. 颗粒样品15分钟。在桌面离心机中为16,100 x g
    2. 小心地清除大部分上清液。
    3. 重新离心样品,并将所有剩余的液体
    4. 空气干燥颗粒 注意:不要颠倒管,药丸是松散的。
    5. 将样品重悬于含有4mM DTT的SDS-PAGE样品缓冲液中
    6. 在100℃加热5分钟
    7. 在样品制备时使用A <600>测量在凝胶上加载当量
    8. 加载每个 -/+对彼此相邻。
    9. 运行凝胶和Western印迹。

  • II。硫酯键断裂后半胱氨酸对烷基化剂的可接近性

    按照A中所述的协议

    1. HA治疗
      1. 在台式离心机中以16,100×g离心1.6ml细胞培养物1分钟。
      2. 将细胞重悬在1ml PB中。
      3. 在标记有 - 和+ HA和离心细胞的管中分开2×500μl
      4. 通过向上和向下移液,将细胞重悬在500μl1M Tris-HCl(pH 7.0),1%SDS( - )或500μl1M HA(pH 7.4),1%SDS 注意:这会使蛋白质变性,并在一个步骤中裂解硫酯键。
      5. 在室温下孵育30分钟。
      6. 加入50μl100%TCA,涡旋
      7. 在冰上孵育30分钟。预冷台式离心机
      8. 在4℃下在16,100×g离心15分钟蛋白质沉淀
      9. 用1ml冰冷的丙酮(储存在-20°C)洗涤沉淀3次,每次以16,100×g离心10分钟。
      10. 空气干燥颗粒。按照E和F中的步骤操作。
        注意:TCA丸粒通常难以溶解。严谨,频繁地涡旋。

    代表数据


    图1.Ep的载脂蛋白N-酰基转移酶(Lnt)的malPEG烷基化谱的实施例。大肠杆菌。半胱氨酸残基23和62被丙氨酸取代,留下单个活性位点半胱氨酸残基C387。 Lnt通过C387在体内形成硫酯酰基 - 酶中间体,并且被阻断用于与malPEG的反应。在用羟胺(HA)处理细胞时,硫酯键被切割,留下C387的游离硫醇基并通过malPEG烷基化。有代表性的例子,请参见Gélis-Jeanvoine 等人(2015)。

    笔记

    结果可能根据所研究的膜蛋白而变化。强烈建议包括阴性和阳性对照以验证手术的技术细节。例如,推荐使用空载体,目的蛋白的无半胱氨酸变体和具有取代的半胱氨酸残基的无活性突变体。

    食谱

    1. 磷酸盐缓冲液(PB)(50mM,pH 7.0)
      制备100毫升1M KH 2 PO 4 4/
      制备100毫升1M K 2 HPO 4
      将1.5375ml K 2 HPO 4与0.9625ml KH 2 PO 4 PO 4混合,并将蒸馏的无菌水加入到50ml
      最终浓度为50mM KPi(pH 7.0)
    2. 0.25 M EDTA
      将93.05g EDTA加入到800ml H 2 O中 用NaOH调节pH至8.0 注意:EDTA二钠不溶于溶液,直到pH约为8.0。
      分成等份和高压灭菌
    3. MTSES溶液(使用前清新)
      对于每个36.3mg,加入500μlH 2 O,最终浓度为0.3M/dm 2
    4. 1μML-半胱氨酸 在1ml H 2 O中的121mg L-半胱氨酸
    5. 95%MeOH
      向47.5ml MeOH中加入H 2 O至50ml
    6. 1M Tris。 HCl
      将121.1g Trisma碱加入到800ml H 2 O中,用HCl调节pH至7.0 将H <2> 添加到1L的最终体积中。
      高压灭菌器
    7. 变性缓冲液(使用前清新)
      为了获得H 2 O:MeOH:CHCl 3 = 1:4:1的溶液,加入到15ml Falcon管中:
      2ml H 2 O 2 / 8ml MeOH
      2ml CHCl 3/
    8. PEG缓冲液
      含有10M尿素,1%SDS,1mM EDTA的1M Tris pH7.0。
      每ml溶液:
      0.6克尿素
      0.01克SDS
      溶解在600μl1M Tris(pH7.0)中,需要混合并浸在温水中
      制备后不要将溶液放在冰上,因为尿素会沉淀
      加入4μl0.25M EDTA
      用1M Tris(pH 7.0)将体积调节至1 ml,
      注意:PEG缓冲液含有尿素,SDS和EDTA,可使所有蛋白质变性,并使所有未受保护的半胱氨酸残基进入malPEG。
    9. malPEG溶液
      注意:使用前请清新,不能储存。
      允许malPEG达到室温
      称量少量,约5 mg,在称量纸上
      添加H 2 O以达到1.2mM的最终浓度MalPEG:对于1mg的malPEG,添加170μl的H 2 O 2。
      注意:对于每个样品,需要10μl的1.2mM malPEG,其对应于12nmol malPEG。 1nmol malPEG为5μg。准确称量的malPEG的最小量为4-5mg。这足以容纳34个样本。
    10. 羟胺溶液(HA)
      将1.4g HA溶解在10ml冷H 2 O中,制成20ml最终的HA溶液。
      用32%NaOH将pH调节至7.4,如下
      加入1.5ml 32%NaOH,搅拌并检查pH值
      逐渐加入更多的NaOH,并检查pH,直到pH为7.4 用H 2 O将体积加至20ml 注意:pH值应在6.9和7.5之间,在pH8.5左右,溶液的特异性会降低。
    11. 1M Tris缓冲液,1%SDS洗涤 加20%SDS至1M Tris溶液至终浓度为1%
    12. SDS-样品缓冲液(4x)
      2.5ml 1M Tris(pH 6.8)
      1.0克SDS
      4ml 100%甘油 0.8ml 0.1%溴酚蓝
      0.16ml 1M DTT
      用dH 2 O
    13. 1 M DTT
      将1.5g DTT溶解在8ml H 2 O中,将体积调节至10ml
      分成1ml等分试样,并储存在-20℃下

    致谢

    这项工作由巴斯德研究所资助。

    参考文献

    1. Bogdanov,M.,Zhang,W.,Xie,J.and Dowhan,W。(2005)。 通过取代的半胱氨酸可接近性方法(SCAM TM)的跨膜蛋白质拓扑学绘图:应用于脂质 - 特异性膜蛋白质形成。方法 36(2):148-171
    2. Gélis-Jeanvoine,S.,Lory,S.,Oberto,J.and Buddelmeijer,N.(2015)。 位于膜嵌入的柔性环上的残基对于载脂蛋白N-酰基转移酶反应的第二步是必需的 。 Mol Microbiol 95(4):692-705。
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    引用:Gélis-Jeanvoine, S. and Buddelmeijer, N. (2015). Substituted Cysteine Accessibility Method for Topology and Activity Studies of Membrane Enzymes Forming Thioester Acyl Intermediates in Bacteria. Bio-protocol 5(21): e1640. DOI: 10.21769/BioProtoc.1640.
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