通过蓝色天然凝胶电泳分析类囊体膜蛋白复合物

摘要

一种利用蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）和二维SDS-PAGE解析植物类囊体蛋白复合物结构与组成的实验方案被详细描述。该方案已针对 拟南芥, 但经过少量修改后也可用于其他植物物种。

摘要

光合电子传递链（ETC）将太阳能转化为NADPH和ATP形式的化学能。嵌入类囊体膜中的四个大型蛋白复合物利用太阳能，通过两个光系统将电子从水传递至NADP⁺，并利用所产生的质子梯度合成ATP。光系统PSII、PSI、细胞色素b₆f（Cyt b₆f）和ATP合酶均为多亚基蛋白复合物，在类囊体膜中具有特定的空间取向和动态行为。通过温和去垢剂将这些复合物从保持完整性的膜中溶解出来，再经非变性凝胶电泳分离，可获得有关类囊体膜中蛋白复合物组成及其相互作用的有价值信息。蓝色非变性聚丙烯酰胺凝胶电泳（BN-PAGE）是一种用于在天然且有功能的状态下分离蛋白复合物的分析方法。该方法既可用于蛋白复合物的纯化以进行更深入的结构分析，也可作为研究蛋白复合物间动态相互作用的工具。该技术最初为线粒体呼吸链蛋白复合物分析而开发，现已针对类囊体蛋白复合物的研究进行了优化和改进。本文提供了一个详细且最新的实验方案，用于分析拟南芥（Arabidopsis thaliana）中不稳定的光合蛋白复合物及其相互作用。

引言

大型多亚基蛋白复合物光系统PSI和PSII、细胞色素b₆f以及ATP合酶共同协调光合作用光反应中NADPH和ATP的生成。在高等植物叶绿体中，这些复合物位于类囊体膜上，该膜是一种结构异质性的膜系统，由堆叠的基粒类囊体和非堆叠的基质类囊体组成。蓝色天然聚丙烯酰胺凝胶电泳（BN-PAGE）是一种广泛用于分析大型多亚基蛋白复合物天然且具有生物学活性形式的方法。该方法最初建立用于线粒体膜蛋白复合物的解析¹，随后被优化用于分离类囊体膜网络中的蛋白复合物³。该方法适用于：（i）纯化单个类囊体蛋白复合物以进行结构分析；（ii）确定蛋白复合物之间的天然相互作用；以及（iii）分析在不同环境信号变化下蛋白复合物整体组织结构的变化。

在分离之前，需使用精心选择的非离子型去垢剂从膜中分离蛋白质复合物，这类去垢剂通常较为温和，能够保持蛋白质复合物的天然结构。去垢剂含有疏水和亲水位点，在达到一定浓度（称为临界胶束浓度，CMC）以上时可形成稳定的胶束。当去垢剂浓度超过CMC时，会破坏脂质-脂质相互作用，从而使蛋白质复合物溶解。去垢剂的选择取决于目标蛋白质复合物的稳定性以及去垢剂的溶解能力。常规使用的去垢剂包括α/β-十二烷基麦芽糖苷（α/β-dodecyl-maltoside）和皂苷（digitonin）。在保持蛋白质复合物天然状态的前提下完成溶解后，通过离心去除不溶性物质。在高等植物中，类囊体膜在结构上具有高度异质性，某些去垢剂（例如，digitonin）仅选择性地溶解膜的特定部分³。因此，为了表征蛋白质复合物的组织结构或复合物之间的相互作用，必须通过测定上清液中的叶绿素含量以及上清液与沉淀中叶绿素a/b比值，来评估所选去垢剂的溶解能力，分别反映可溶部分的得率及其代表的类囊体（亚）区域。在适应生长光条件的植物中，完整类囊体的叶绿素a/b比值通常约为3，而富含基粒或基质类囊体的类囊体组分，其叶绿素a/b比值则分别低于（约2.5）或高于（约4.5）总类囊体的该比值。

为了给蛋白质复合物提供负电荷，将考马斯亮蓝（CBB）染料加入溶解后的样品中。由于电荷的改变，蛋白质复合物向阳极迁移，并根据其分子质量和形状在丙烯酰胺（AA）梯度凝胶中实现分离。通过使用线性丙烯酰胺浓度梯度，可实现高效且高分辨率的分离。在电泳过程中，蛋白质复合物向阳极迁移，直至达到与其大小相关的孔径极限。聚丙烯酰胺凝胶的孔径大小取决于（i）总丙烯酰胺/bis-丙烯酰胺浓度（T）以及（ii）交联剂bis-丙烯酰胺单体浓度（C）相对于总单体的比例⁴。在通过BN-PAGE完成分离后，可通过第二维（2D）SDS-PAGE将蛋白质复合物进一步分离为其各自的蛋白质亚基。本文详细描述了利用BN-PAGE/2D-SDS-PAGE分析类囊体膜蛋白质复合物的实验方案。

方案

1. Preparing BN Gel^1,2,3

1. Set up the gel caster with 8 cm x 10 cm plates (rectangular glass and notched alumina plate) according to manufacturer's instructions using 0.75 mm spacers.
2. Place a gradient mixer on a stir plate and connect it with the peristaltic pump by a tubing. Attach a syringe needle to the other end of the tubing and place the needle between the glass and aluminum plate. Place magnetic stirrer to the "heavy" (H)-chamber.
3. Prepare the 3.5% (v/v) and 12.5% (v/v) acrylamide (AA) solutions in 15 mL conical centrifuge tubes for the separation gel gradient (see recipes in Table 1). To prevent untimely polymerization, keep the centrifuge tubes on ice while preparing the solutions.
   CAUTION: Acrylamide is neurotoxic and carcinogenic, wear protective clothes and gloves.
4. Add 5% APS and TEMED right before pipetting the solutions to the gradient mixer. Pipet the 12.5% solution to the H-chamber.
   1. Remove air bubbles from the channel connecting the "light" (L) and H-chamber by opening the valve connecting the two chambers allowing solution to enter to the L-chamber. Close the valve and pipet the traces of solution back to H-chamber.
   2. Finally, pipet the 3.5% solution to the L-chamber.
5. Switch on the magnetic stirrer (the speed of the stir is not critical, but it should ensure proper mixing of the heavy and light solutions), open the valves and switch on the peristaltic pump. Allow the gel solutions to flow between the glass and aluminum plate, the flowrate should be roughly 0.5 mL/min. The needle must be above the liquid all the time, it can be attached to the upper part of the glass plate with a tape.
6. When the H- and L-chambers have emptied, fill them with ultrapure water and allow water to gently overlay the gel surface. The gel polymerization takes around 1-2 hours at RT.
7. Prepare the 3% acrylamide solution (see recipe in Table 1) for the stacking gel at RT. Pipet the stacking gel on top of the polymerized separation gel (before casting the stacking gel, remove the water overlaying the gel surface) and place a sample gel comb between the glass and aluminum plate avoiding air bubbles.
   1. Allow to polymerize 30-60 min at RT. Remove the comb gently under ultrapure water. Store the gel at +4 ˚C.
      Pause point. The gel can be stored at +4 ˚C for a few days. The gel should be kept in moist conditions to avoid drying of the wells and the gel surface.

2. Thylakoid Solubilization^1,2,3

NOTE: All steps should be performed under very dim light. Keep samples and buffers on ice.

1. Dilute isolated thylakoids with ice-cold 25BTH20G buffer to a final chlorophyll concentration of 1 mg/mL. For 2D-BN-SDS-PAGE analysis roughly 4-8 µg of chlorophyll/ sample is suitable.
   NOTE: The thylakoids used in the experiments must be isolated from fresh leaves (for protocol of thylakoid isolation, see³)
2. Add an equal volume of detergent buffer, i.e., 2% β-DM (w/v) or 2% digitonin (w/v). Mix the detergent to the thylakoid sample gently with the pipet tip and avoid making air bubbles. The final concentration of the detergent is 1% and that of the thylakoids 0.5 mg Chl/mL.
   1. Solubilize the thylakoids for 2 min on ice (β-DM) or 10 minutes at RT with continuous gentle mixing on a rocker/shaker (digitonin).
      NOTE: Digitonin and β-DM are generally used for the solubilization of thylakoid protein complexes. If other non-ionic detergents are used, the detergent concentration and the solubilization time must be first optimized. Usually the detergent concentration range from 0.5%-5% (v/v).
      CAUTION: Digitonin is toxic, wear protective clothes and gloves
3. Remove the insolubilized material by centrifugation at 18,000 x g for 20 min, at +4 ˚C.
4. Transfer the supernatant to a new 1.5 mL tube and add 1/10 (v/v) of CBB buffer to the sample.
   NOTE: When the overall composition of the thylakoid membrane protein complexes is examined, determining the yield and the represented thylakoid domain of solubilized fraction is recommended. To determine the yield of the solubilized material, take 5 µL of the supernatant to a new tube (before adding CBB) and measure the Chl content and Chl a/b ratio⁵.

3. BN-PAGE^1,2,3

1. Set up the gel to a vertical electrophoresis system (e.g., Hoefer SE 250). Fill the upper buffer chamber with blue cathode buffer (see Table 1) and pour anode buffer to the lower buffer chamber.
2. Load thylakoid sample (e.g., 5 µg of chlorophyll) into the wells.
3. Start the electrophoresis and gradually increase the voltage: 75 V for 30 min, 100 V for 30 min, 125 V for 30 min, 150 V for 1 h and 175 V until the complexes have been separated completely. Run the gel at +4˚C, either using a cold room or adjusting the gel temperature with a cooling system.
   NOTE: Change the blue cathode buffer to a clear cathode buffer when the sample front has migrated about one third of the gel.
4. After the electrophoretic run, scan the gel with a photoscanner for image archiving.

4. 2D-SDS-PAGE

1. Assemble the vertical electrophoresis system (gel size 16 cm x 20 cm). Use 1 mm spacers.
2. Prepare standard SDS gel (12% acrylamide, 6 M Urea, see recipe in Table 2) with a 2D-comb (single large well for the strip and one standard well for molecular weight marker).
3. Cut the lane from BN-gel and place it in a (5 mL) tube. Add 2 mL of Laemmli buffer (containing 5% β-mercaptoethanol) and incubate the strip for 45 min with gentle shaking at RT.
4. Place the lane, with a help of e.g., a spacer, on top of the gel avoiding air bubbles.
5. Pipet 5 µL of molecular weight marker on a narrow piece of filter paper and place the paper to the standard well.
6. To seal the BN-gel strip and the marker paper, pour 0.5% agarose (in running buffer) on top of the gel strip and allow to solidify.
7. Perform electrophoresis according to standard protocols. After the electrophoretic run, visualize the proteins with e.g., Sypro Ruby stain or silver staining according to⁶.

结果

一种具有代表性的二维氮化硼/SDS-聚丙烯酰胺凝胶电泳系统 图1 展示了经皂苷素（digitonin）和β-DM溶解的类囊体蛋白复合物的分离及其蛋白质亚基组成的详细分析。皂苷素溶解类囊体的蛋白复合物图谱（上方水平胶图） 图1A包含PSII-LHCII-PSI巨复合体、两个大型PSII-LHCII超复合体（sc）、PSI-LHCII超复合体、PSI单体（m）以及PSII m/Cyt b₆f，松散结合的（L）-LHCII三聚体（图1A）。稍强的去垢剂β-DM可溶解整个类囊体膜（顶部的水平凝胶） 图1B)，但无法保留蛋白质复合物之间的弱相互作用。使用β-DM溶解类囊体通常会产生四种PSII-LHCII超级复合物（结合不同数量的LHCII天线）、PSII二聚体（d）和PSI单体、ATP酶、PSII单体以及细胞色素b

讨论

光合作用能量转换系统由嵌入类囊体膜中的大型多亚基蛋白复合物组成。本方案描述了一种利用非变性凝胶电泳（BN-PAGE）结合二维SDS-PAGE分析拟南芥（Arabidopsis thaliana）类囊体蛋白复合物的基本方法。该方案也适用于烟草和菠菜类囊体蛋白复合物的分析，但可能需要进行少量调整。

为了溶解膜蛋白复合物，通常使用非离子型去污剂，因其能够保持复合物的天然构象。本研究中采用了两种常用的去污剂：β-DM 和皂苷（digitonin）。十二烷基麦芽糖苷（dodecyl maltoside）可溶解单个蛋白复合物，而皂苷则适用于较大蛋白复合物组装体的分析¹⁰。由于皂苷分子结构较大，无法进入紧密堆叠的基粒类囊体区域，因此仅能溶解类囊体膜中非堆叠区域^3,11。这使其适用于基质类囊体和基粒边缘区域的分析。然而，当皂苷与氨基己酸（aminocaproic acid, ACA）联合使用时，该组合可溶解整个类囊体...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
6-氨基己酸（ACA）	Sigma-Aldrich	A2504
BisTris	Sigma-Aldrich	B4429
蔗糖	Sigma-Aldrich	S0389
丙烯酰胺（AA）	Sigma-Aldrich	A9099	注意：具有神经毒性！
n-十二烷基-β-D-麦芽糖苷	Sigma-Aldrich	D4641
Tricine	Sigma-Aldrich	T0377
Tris	Sigma-Aldrich	T1503
SDS	VWR	442444H
尿素	VWR	28877.292
甘油	J.T. Baker	7044
氟化钠（NaF）	J.T. Baker	3688
EDTA 二钠盐	J.T. Baker	1073
皂苷	Calbiochem	300410	注意：有毒！
Pefabloc SC	Roche	11585916001
Serva 考马斯亮蓝 G	Serva	35050
β-巯基乙醇	Bio-Rad	1610710
APS（过硫酸铵）	Bio-Rad	161-0700
TEMED（四甲基乙二胺）	Bio-Rad	1610801
（N,N'-亚甲基）双丙烯酰胺	Omnipur	2610
甘氨酸	Fisher	G0800
预染蛋白 Marker，宽范围（7–175 kDa）	New England Biolabs	P7708
Falcon 15 ml 锥形离心管	Corning	352093
双凝胶铸模装置，配有 10 × 8 cm 板	Hoefer	SE215
梯度混合器 SG5	Hoefer
0.75 mm T 型间隔片	Hoefer	SE2119T-2-.75
样品胶梳，0.75 mm	Hoefer	SE211A-10-.75
Mighty Small SE250 垂直电泳系统	Hoefer	SE250
IPC 泵	Ismatec
电源，PowerPac HV	Bio-Rad	164-5097
离心机	Eppendorf	5424R
摇床-振荡器	Biosan	BS-010130-AAI
PROTEAN II xi Cell	Bio-Rad	1651813