Letter to the Editor: Weak-Acidic Clear-Native Polyacrylamide Gel Electrophoresis for the Separation of the Intact Forms of Thylakoid Protein Complexes
Letter to the Editor: Weak-Acidic Clear-Native Polyacrylamide Gel Electrophoresis for the Separation of the Intact Forms of Thylakoid Protein Complexes
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致编辑的信：弱酸性透明天然聚丙烯酰胺凝胶电泳用于分离完整形式的类囊体蛋白复合物
DOI：
10.1093/pcp/pcac070
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发表时间：
2022
影响因子：
4.9
通讯作者：
Takabayashi Atsushi
中科院分区：
文献类型：
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作者：
Matsumae Renon;Kameo Shinsa;Tanaka Ryouichi;Takabayashi Atsushi
蓝色-天然（BN）-聚丙烯酰胺凝胶电泳（PAGE）和透明-天然（CN）-PAGE是分离光合机构组分的常用技术。这些技术的一个局限性是，弱碱性运行pH值（pH 7.5; Schägger和von Jagow 1991）会导致析氧复合物从光系统II中解离（PSII; Crepin等人，2016）。由于离子（如前导离子、拖尾离子和缓冲液离子）的迁移，运行pH值随缓冲液pH值（pH 7.0）变化。在此，我们交流了一种新的CNPAGE系统，称为弱酸性（wa）CN-PAGE（图1A;详见补充方法），它规避了这一警告。在新的waCN-PAGE系统中，将阴极、阳极和凝胶缓冲液的pH调节至6.5。我们取代tricine（8.15 pKa），在传统的CN-PAGE，2-吗啉代乙磺酸（6.15 pKa）作为尾随离子。拖尾离子在电泳过程中应具有部分负电荷;然而，在弱酸性条件下tricine的阴离子形式不足以作为waCN-PAGE的拖尾离子。此外，我们测试了两种不同的缓冲离子，BisTris和组氨酸，在电泳过程中稳定凝胶的pH值。我们还使用amphipol A8 - 35代替广泛使用的脱氧胆酸钠，在电泳过程中为蛋白质复合物赋予负电荷，因为已知amphipol A8 - 35可防止LHCII解离（Kameo等人，2021）。有关缓冲系统的其他信息，请参见补充结果和讨论。作为概念证明，通过常规CN-PAGE和通过我们的waCN-PAGE系统（使用组氨酸作为缓冲离子）解析光系统的大复合物（PS大复合物）、PSII-LHCII超复合物、PSI-LHCI和LHCII三聚体（图1B）。我们发现，用常规CN-PAGE分离后，凝胶上叶绿体（cp）ATP合酶亚基斑点（AtpA、AtpB和AtpC）的模式与用waCN-PAGE分离后观察到的模式有很大不同（图1C、D）。除了接近PSI-LHCI的完全组装的cpATP合酶（Rühle et al. 2014）外，与银染2D-waCN/SDS-PAGE凝胶（图1D）相比，银染2D-CN/SDS-PAGE凝胶（图1C）中对应于较小cpATP合酶的斑点更明显，如使用抗AtpA抗体的免疫印迹分析所证实的（图1C、D）。这些较小的斑点可能代表cpATP合酶复合物的解离亚基，表明该复合物在waCN-PAGE中似乎比在CN-PAGE凝胶中更稳定。我们还在两种系统下检查了PSII-LHCII超复合物，发现使用抗PsbP抗体的免疫印迹分析表明，在常规CN-PAGE系统中，PsbP从PSII-LHCII超复合物中解离（图1C）。相比之下，PsbP保留在waCN-PAGE系统中的PS大复合物和PSII-LHCII超复合物内（图1D）。考虑到PsbP的分子量（23 kDa）和免疫印迹分析中获得的条带模式，常规CN-PAGE凝胶（图1C）上低分子量区域中的宽拖尾条带（在2D-waCN/SDS-PAGE凝胶（图1D）上未观察到）可能对应于PsbP。这些发现表明在waCN-PAGE系统中有效地防止了PsbP从PSII-LHCII超复合物解离。此外，我们发现，通过改变缓冲
Blue-native (BN)-polyacrylamide gel electrophoresis (PAGE) and clear-native (CN)-PAGE are commonly used techniques for separating components of the photosynthetic machinery. Alimitationofthesetechniquesisthattheslightlybasicrunning pH (pH 7.5; Schägger and von Jagow 1991) causes oxygenevolvingcomplextodissociatefromphotosystemII (PSII; Crepin et al. 2016). The running pH changes with the pH of buffers (pH 7.0) due to the migration of ions such as leading, trailing, and buffer ions. Herein, we communicate a new CNPAGE system, termed weak-acidic (wa) CN-PAGE (Fig. 1A; see Supplementary methods for details) that circumvents this caveat. In the new waCN-PAGE system, the pH of the cathode, anode, and gel buffers was adjusted to 6.5. We substituted tricine (8.15 pKa), used in conventional CN-PAGE, for 2-morpholinoethanesulfonic acid (6.15 pKa) as the trailing ion. A trailing ion should have a partial negative charge during electrophoresis; however, the anionic form of tricine under weak-acidic condition is not sufficient as the trailing ion of waCN-PAGE. In addition, we tested two different buffering ions, BisTris and histidine, that stabilize the pH of the gel during electrophoresis. We also used amphipol A8-35 instead of the widely used sodium deoxycholate to confer negative charges to the protein complexes during electrophoresis because amphipol A8-35 is known to prevent LHCII dissociation (Kameo et al. 2021). Additional information on the buffer system is described in the Supplementary Results and Discussion. As proof of concept, the megacomplexes of photosystems (PS megacomplexes), PSII-LHCII supercomplexes, PSI-LHCI and LHCII trimers were resolved through conventional CN-PAGE and through our waCN-PAGE system using histidine as the buffering ion (Fig. 1B). We found that the patterns of chloroplast (cp) ATP synthase subunit spots (AtpA, AtpB and AtpC) on the gel after separation by conventional CN-PAGE were considerably different from those observed after separation by waCN-PAGE (Fig. 1C, D). In addition to the fully assembled cpATP synthase close to PSI-LHCI (Rühle et al. 2014), spots corresponding to smaller cpATP synthase were more notably in the silver-stained 2D-CN/SDS-PAGE gel (Fig. 1C) than in the silver-stained 2D-waCN/SDS-PAGE gel (Fig. 1D), as corroborated by immunoblot analysis using anti-AtpA antibodies (Fig. 1C, D). These smaller spots likely represent dissociated subunits of the cpATP synthase complex, indicating that this complex appears to be more stable in waCN-PAGE than in CN-PAGE gels.We also checked the PSII-LHCII supercomplex under both systems and found that immunoblot analysis using anti-PsbP antibodies demonstrated that PsbP was dissociated from the PSII-LHCII supercomplex in the conventional CN-PAGE system (Fig. 1C). In contrast, PsbP was retained within the PS megacomplexes and the PSII-LHCII supercomplex in the waCN-PAGE system (Fig. 1D). Considering the molecular weight of PsbP (23 kDa) and band pattern obtained in the immunoblot analysis, a broad tailing band in the low molecular weight region on the conventional CN-PAGE gel (Fig. 1C), which was not observed on the 2D-waCN/SDS-PAGE gel (Fig. 1D), likely corresponds to PsbP. These findings demonstrate that PsbP dissociation from the PSII-LHCII supercomplex was effectively prevented in the waCN-PAGE system. Furthermore, we found that by changing the buffering