类囊脂质体中硅藻Cyclotella Meneghiniana中的光捕获天线的分离与融合
Summary
在这里, 我们提出了一种从硅藻中分离 fucoxanthin 叶绿素 a/c 结合蛋白 (FCP) 的协议, 并将其纳入脂质体和天然脂质成分, 研究了离子成分变化时的激发能量传递。
Abstract
植物、藻类和硅藻的光合性能强烈依赖于叶绿体类囊体膜中的光收获和能量传递过程的快速有效调控。硅藻的光收获天线, 所谓的 fucoxanthin 叶绿素 a/c 结合蛋白 (FCP), 是需要的光吸收和有效地转移到光合反应中心, 以及为光保护从过量的光。这两个函数之间的切换是一个长期的研究课题。其中许多研究都是用 FCP 在洗涤剂胶束中进行的。在相互作用研究中, 洗涤剂被去除, 导致 FCP 复合物的不特异聚集。在这种方法中, 很难区分工件和生理相关数据。因此, 通过研究蛋白质-蛋白质相互作用, 能量转移和其他光谱特征, 如果它们嵌入在他们的本地脂质环境中, 就可以获得更有价值的 FCP 和其他膜结合光采集配合物的信息。其主要优点是脂质体具有定义的大小和定义的脂/蛋白比, FCP 聚类的程度受到控制。此外, 在体内调节光收获的 pH 值和离子成分的变化也很容易被模拟。与类囊膜相比, 脂质体更均匀、不复杂, 便于获得和理解光谱数据。该协议描述了 FCP 分离纯化、脂质体制备、FCP 脂质体与天然油脂成分结合的过程。给出了典型应用的结果, 并进行了讨论。
Introduction
像硅藻这样的光合有机体必须应对不断变化的光照条件, 并应对复杂的适应机制, 保持高光合效率和保护光氧化损伤所造成的过度光照。光合真核生物的一个主要的光保护过程是高能淬火 (qE) 的吸收光作为主要贡献的非光化学淬火 (NPQ) 在轻应力条件下1,2 ,3。光采集天线配合物 (LHC) 参与了励磁能量传递通路的调节。由于在叶绿体腔内高光诱导低pH 值, 天线系统从光收获状态切换到淬火状态。这种能量耗散状态保护-高等植物 (PS) 和其他配合物在类囊体膜从光氧化。在光合真核生物中, qE通常由两个因素1、2、3引起。一个因素是专门的光收获蛋白对低pH 值的反应。公安局蛋白在高等植物中诱导 qE 4。LhcSRs5, 由公安局活动调制, 诱导绿藻中 qE 6。硅藻具有与 LHCSRs7、8、9、10的结构相关的 Lhcx 蛋白。
qE的第二个因素是叶黄素的周期, 其中的类胡萝卜素被转化成光保护形式, 通过环氧化和还原环氧化。在植物和绿藻中, 紫黄素转化为玉米黄质。在硅藻中, diadinoxanthin 转换为 diatoxanthin, 然后与 NPQ11的程度相关。硅藻光收获天线具有一定的特殊性, 尽管它与植物和藻类 LHCs 有关。从光照到光保护的转换速度非常快, 与植物12相比, NPQ 容量更高。这可能是为什么硅藻在不同的生态环境中非常成功的原因之一, 他们负责45% 的大洋净初级产量13。因此, 硅藻光收获系统是光合作用研究的一个有趣的对象。
硅藻, 像中心物种Cyclotella meneghiniana,拥有类囊体固有的光采集系统, 命名后的颜料, 他们绑定-fucoxanthin, 叶绿素 (智利) a 和 c, 因此 FCP. 光收获蛋白, 如 FCPs, 是嵌入在囊体膜系统中, 包括几个膜层。硅藻形成三类囊体的带状。这种复杂的情况使得研究它们在类囊体膜中的分子水平是困难的。此外, 许多组件有助于调节光的收获 (见上文)。因此, 在许多方法中, 复合体是从膜中分离出来的, 使用温和的洗涤剂, 如 n-月桂-maltopyranoside (β), 溶解膜, 但保持 FCP 配合完好。利用可溶性 FCP 对分子内能量转移14、15、16、17进行了多光谱研究。然而, 这种前一种方法是有限的, 因为调节能量转移需要激子吸收与其他天线配合物或-高等植物的相互作用。因此, 这些类型的研究不能与可溶性配合, 因为复合体之间的相互作用失去了。
天线调节的一个重要特征是类囊体膜18中天线和-高等植物的 “分子拥挤”。以前, 一种简单的方法来模拟这种效应的体外.洗涤剂被去除, 导致天线复合体的随机聚集。虽然这一方法17、19获得了一些合理的数据, 但洗涤剂的去除并没有反映体内的情况, 也有一些局限性, 因为复合物在其正规的第三系中没有相互作用。结构。
脂质体的使用克服了前几个局限性。第三级结构仍然完全完好。脂质体膜为天线配合物提供了准本机环境。膜从外部环境中分离脂质体的内部。通过这些方法, 脂质体提供了两个反应室, 用于研究离子和 pH 梯度以及运输过程。此外, 实验系统的参数可以更容易地控制在类囊体膜的研究。脂质体已经被证明是研究光合络合物的极好工具。过去的一个主要重点是植物 lhc, 在那里, 改变脂质成分的影响测试了 LHC II20。在其他方法中, 研究了不同的 LHC II. 的蛋白质-蛋白质相互作用21。此外, 还对绿藻进行了一些研究, 描述了 LHC22之间的自发聚类。考虑到硅藻在水生生态系统中的重要性, 对硅藻天线配合物进行了比较少的研究。两项研究调查了中心Cyclotella meneghiniana的天线配合物, 其中显示了 FCP 天线23的聚类和 FCP 对电化学梯度24的响应。因此, 脂质体是研究硅藻天线及其在近地环境中相互作用和调节的极好工具。脂质体是多才多艺的, 因为许多条件, 如脂组成, 脂质体的大小, 蛋白质密度和周围的水相可以控制。此外, 该方法需要少量的样本。实验系统鲁棒性强, 重现性高。脂质体的划分允许研究 pH 值和离子梯度, 这是调节天线配合物的重要因素。
在这里, 我们描述了 FCP 天线配合物的分离从C. meneghiniana和他们的纳入脂质体与自然类囊脂组成。同时, 我们为可溶性 FCP 的光谱表征提供了典范的数据, 并与脂质体中的 FCP 进行了比较。该方法总结了从改进 Gundermann 和 Büchel 201223、娜塔莉等201622、艾哈迈德和 Dietzel 201724获得的知识和标准化协议。
图 1: 工作流的示意图表示形式。(1)是指1段, 描述细胞生长, 破坏和类囊体的分离与以下 FCP 分离的蔗糖密度梯度;沙菲·萨米–Cyclotella meneghiniana细胞。(2) 2 段中描述的天然类囊体脂质混合物 (MGDG、DGDG 和 SQDG) 的制备, 并建立 octylglycoside 脂质洗涤剂胶束。所定义的脂胶束大小是通过使用已定义孔径的膜挤压而实现的。FCP 和脂胶束在预定的脂质上统一: 蛋白质比,通过控制透析形成 FCP proteoliposomes 去除清洁剂和β。请单击此处查看此图的较大版本.
Protocol
Representative Results
Discussion
FCP 脂质体具有天然脂质成分, 为研究其体外光谱特性提供了方便、简便、重现性的工具。FCP 脂质体的脂质环境类似于类囊膜内的情况, 导致实验结果更接近自然条件。
利用meneghiniana作为 FCP 天线的模型系统具有多种优点。它的生长相对较快, 比其他硅藻模型物种更健壮,例如海链藻 pseudonana。在珠磨中, 细胞断裂相对容易, 这使得完整的囊状体复合物的高产。…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢阿迪尔. 艾哈迈德在 FCP 净化方面的帮助。克劳迪娅教授 Büchel 被公认为有帮助的讨论和阅读手稿。这项工作得到德国研究基金会 (DI1956-1/1) 和洪堡基金会 Feodor Lynen 奖学金的支持。
Materials
| 500 ml centrifuge vials | |||
| high speed centrifuge | Heraeus | ||
| Bead Mill VI 2 | Edmund-Bühler (edmund-buehler.de) | newer version: Vibrogen-Zellmühle Vl 6 | |
| Silibeads S 400 µm | Sigmund-Lindner.com | 5223-7 | |
| Silibeads S 1,-1,3 mm | Sigmund-Lindner.com | 4504 | |
| VitraPOR filter funnel – por1 | ROBU GmbH | 21121 | |
| polycarbonate ultracentrifuagtion vials (30 mL) for T-865 | Beranek Laborgeräte (Laborgeraete-beranek.de) | 314348 | |
| Ultracentrifuge Discovery 90SE | Sorvall | n.a. | |
| rotor T 865 | ThermoFisher Scientific (thermofisher.com) | 51411 | |
| Neubauer Cell Counter Chamber (improved) | Carl Roth Laborbedarf (Carlroth.com) | T729.1 | |
| Zeiss Mikroskop Primostar (7) | Optik-Pro (optik-pro.de) | 51428 | |
| optical glass cuvettes (6040-OG) | Hellma Analytics (hellma-analytics.com) | "6040-10-10" | |
| V-630 UV-VIS Spectrophotometer (incl. software) | Jasco (jasco.de) | V-630 | |
| n-Dodecyl-β-D-Maltopyranoside | ANATRACE (anatrace.com) | D310LA | |
| Ultra-Clear tubes 17 ml for AH629 | Beranek Laborgeräte (Laborgeraete-beranek.de) | 344061 | |
| rotor AH629-17-mL | ThermoFisher Scientific (thermofisher.com) | 54285 | |
| Membrane concentrator_Centriprep 30 kDa cutoff | Millipore (merckmillipore.com) | 4307 | |
| Biometra Minigel-Twin | Analytik Jena AG (analytik-jena.de) | 846-010-100 | |
| Silver Stain Plus Kit | Bio-Rad (bio-rad.com) | 1610449 | |
| libre office spread sheet | The document foundation | https://de.libreoffice.org/download/libreoffice-still/ | |
| special glass cuvettes for fluorescence (101-0S) | Hellma Analytics (hellma-analytics.com) | 101-10-20 | |
| Spectrofluorometer FP-6500 (incl. Software) | Jasco (jasco.de) | FP-6500 | |
| SDS-loading buffer Roti-Load | ROTH (carlroth.com) | K929.1 | |
| n-octyl β-D-glucopyranoside | ANATRACE (anatrace.com) | O311 | |
| Monogalactosyl Diaclyglycerol (MGDG) | Larodan AB (larodan.com) | 59-1300 | make stock solution in chloroform |
| Digalactosyl Diacylglycerol (DGDG) | Larodan AB (larodan.com) | 59-1310 | make stock solution in chloroform |
| Sulphoquinovosyl Diacylglycerol (SQDG) | Larodan AB (larodan.com) | 59-1230 | make stock solution in chloroform |
| L-alpha-Phosphatidylglycerol (PG) | Larodan AB (larodan.com) | 37-0150 | make stock solution in chloroform |
| L-α-Phosphatidylcholine | Sigma-Aldrich (sigmaaldrich.com) | P3782 SIGMA | make stock solution in chloroform |
| sonicator bath S-50TH | Sonicor (getmedonline.com | SONICOR-S-50TH | |
| mini-Extruder | Avanti Polar Lipids (Avanti.com) | 610000 | |
| Nuleopore polycarbonate membrane | Avanti Polar Lipids (Avanti.com) | 610005 | |
| dialysis membrane Visking 14 kDa cutoff | ROTH (carlroth.com) | 0653.1 | boil in destilled water before use |
| Biobeads SM2 Adsorbent | Biorad (Bio-rad.com) | 152-3920 | |
| sucrose epichlorhydrin copolymer – Ficoll 400 | Sigma-Aldrich (sigmaaldrich.com) | F4375 | |
| Polycarbonate ultracentrifuagtion vials (2.7 mL) for TFT 80.4 | Beranek Laborgeräte (Laborgeraete-beranek.de) | 252150 | |
| rotor TFT 80.4 | Millipore (merckmillipore.com) | 54356 | |
| material listed in order of appearance | |||
| For specific safety instructions please refer to material safety sheets and repective manuals. | |||
| Standard lab material and substances are not listed. |
References
- Eberhard, S., Finazzi, G., Wollman, F. A. The Dynamics of Photosynthesis. Annual Review of Genetics. 42, 463-515 (2008).
- Li, Z. R., Wakao, S., Fischer, B. B., Niyogi, K. K. Sensing and Responding to Excess Light. Annual Review of Plant Biology. 60, 239-260 (2009).
- Niyogi, K. K., Truong, T. B. Evolution of flexible non-photochemical quenching mechanisms that regulate light harvesting in oxygenic photosynthesis. Current Opinion in Plant Biology. 16 (3), 307-314 (2013).
- Li, X. -. P., et al. A pigment-binding protein essential for regulation of photosynthetic light harvesting. Nature. 403 (6768), 391-395 (2000).
- Peers, G., et al. An ancient light-harvesting protein is critical for the regulation of algal photosynthesis. Nature. 462 (7272), 518-521 (2009).
- Correa-Galvis, V., et al. Photosystem II Subunit PsbS Is Involved in the Induction of LHCSR Protein-dependent Energy Dissipation in Chlamydomonas reinhardtii. The Journal of biological chemistry. 291 (33), 17478-17487 (2016).
- Bailleul, B., et al. An atypical member of the light-harvesting complex stress-related protein family modulates diatom responses to light. Proceedings of the National Academy of Sciences of the United States of America. 107 (42), 18214-18219 (2010).
- Taddei, L., et al. Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum. Journal of experimental botany. 67 (13), 3939-3951 (2016).
- Lepetit, B., et al. The diatom Phaeodactylum tricornutum adjusts nonphotochemical fluorescence quenching capacity in response to dynamic light via fine-tuned Lhcx and xanthophyll cycle pigment synthesis. New Phytologist. 214 (1), 205-218 (2017).
- Büchel, C. Evolution and function of light harvesting proteins. Journal of Plant Physiology. 172, 62-75 (2015).
- Lavaud, J., Rousseau, B., van Gorkom, H. J., Etienne, A. -. L. Influence of the Diadinoxanthin Pool Size on Photoprotection in the Marine Planktonic Diatom Phaeodactylum tricornutum. Plant Physiology. 129 (3), 1398-1406 (2002).
- Ruban, A., et al. The super-excess energy dissipation in diatom algae: comparative analysis with higher plants. Photosynthesis Research. 82 (2), 165-175 (2004).
- Mann, D. G. The species concept in diatoms. Phycologia. 38 (6), 437-495 (1999).
- Papagiannakis, E., van Stokkum, I. H. M., Fey, H., Büchel, C., van Grondelle, R. Spectroscopic Characterization of the Excitation Energy Transfer in the Fucoxanthin-Chlorophyll Protein of Diatoms. Photosynthesis Research. 86 (1-2), 241-250 (2005).
- Premvardhan, L., Robert, B., Beer, A., Büchel, C. Pigment organization in fucoxanthin chlorophyll a/c2 proteins (FCP) based on resonance Raman spectroscgopy and sequence analysis. Biochimica et Biophysica Acta-Bioenergetics. 1797 (9), 1647-1656 (2010).
- Gildenhoff, N., Herz, J., Gundermann, K., Büchel, C., Wachtveitl, J. The excitation energy transfer in the trimeric fucoxanthin-chlorophyll protein from Cyclotella meneghiniana analyzed by polarized transient absorption spectroscopy. Chemical Physics. 373 (1), 104-109 (2010).
- Ramanan, C., et al. Exploring the mechanism(s) of energy dissipation in the light harvesting complex of the photosynthetic algae Cyclotella meneghiniana. Biochimica et Biophysica Acta-Bioenergetics. 1837 (9), 1507-1513 (2014).
- Haferkamp, S., Kirchhoff, H. Significance of molecular crowding in grana membranes of higher plants for light harvesting by photosystem II. Photosynthesis Research. 95 (2-3), 129-134 (2008).
- Wahadoszamen, M., et al. Stark fluorescence spectroscopy reveals two emitting sites in the dissipative state of FCP antennas. Biochimica et Biophysica Acta-Bioenergetics. 1837 (1), 193-200 (2014).
- Zhou, F., et al. Effect of monogalactosyldiacylglycerol on the interaction between photosystem II core complex and its antenna complexes in liposomes of thylakoid lipids. Photosynthesis Research. 99 (3), 185-193 (2009).
- Moya, I., Silvestri, M., Vallon, O., Cinque, G., Bassi, R. Time-resolved fluorescence analysis of the photosystem II antenna proteins in detergent micelles and liposomes. 생화학. 40 (42), 12552-12561 (2001).
- Natali, A., et al. Light-harvesting Complexes (LHCs) Cluster Spontaneously in Membrane Environment Leading to Shortening of Their Excited State Lifetimes. Journal of Biological Chemistry. 291 (32), 16730-16739 (2016).
- Gundermann, K., Büchel, C. Factors determining the fluorescence yield of fucoxanthin-chlorophyll complexes (FCP) involved in non-photochemical quenching in diatoms. Biochimica et Biophysica Acta-Bioenergetics. 1817 (7), 1044-1052 (2012).
- Ahmad, R. A., Dietzel, L. Relaxation of cellular K+ gradients by valinomycin induces diatoxanthin accumulation in Cyclotella meneghiniana cells and alters FCPa fluorescence yield in vitro. Physiologia Plantarum. , 171-180 (2017).
- Provasoli, L., McLaughlin, J. J. A., Droop, M. R. The development of artificial media for marine algae. Archiv für Mikrobiologie. 25 (4), 392-428 (1957).
- Jeffrey, S., Humphrey, G. New spectrophotometry equations for determining chlorophyll a, chlorophyll b, chlorophyll c-1 and chlorophyll c-2 in higher plants, algae and natural phytoplankton. Biochemie und Physiologie der Pflanzen. 167, 191-194 (1975).
- Beer, A., Gundermann, K., Beckmann, J., Büchel, C. Subunit Composition and Pigmentation of Fucoxanthin−Chlorophyll Proteins in Diatoms: Evidence for a Subunit Involved in Diadinoxanthin and Diatoxanthin Binding. 생화학. 45 (43), 13046-13053 (2006).
- Schägger, H., von Jagow, G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry. 166 (2), 368-379 (1987).
- Büchel, C. Fucoxanthin-Chlorophyll Proteins in Diatoms: 18 and 19 kDa Subunits Assemble into Different Oligomeric States. 생화학. 42 (44), 13027-13034 (2003).
- Vieler, A., Wilhelm, C., Goss, R., Süß, R., Schiller, J. The lipid composition of the unicellular green alga Chlamydomonas reinhardtii and the diatom Cyclotella meneghiniana investigated by MALDI-TOF MS and TLC. Chemistry and Physics of Lipids. 150 (2), 143-155 (2007).
- Gundermann, K., Büchel, C. The fluorescence yield of the trimeric fucoxanthin-chlorophyll-protein FCPa in the diatom Cyclotella meneghiniana is dependent on the amount of bound diatoxanthin. Photosynthesis Research. 95 (2-3), 229-235 (2008).
- Miloslavina, Y., et al. Ultrafast fluorescence study on the location and mechanism of non-photochemical quenching in diatoms. Biochimica et Biophysica Acta-Bioenergetics. 1787 (10), 1189-1197 (2009).
- Grouneva, I., Jakob, T., Wilhelm, C., Goss, R. The regulation of xanthophyll cycle activity and of non-photochemical fluorescence quenching by two alternative electron flows in the diatoms Phaeodactylum tricornutum and Cyclotella meneghiniana. Biochimica et Biophysica Acta-Bioenergetics. 1787 (7), 929-938 (2009).
- Chukhutsina, V. U., Büchel, C., van Amerongen, H. Disentangling two non-photochemical quenching processes in Cyclotella meneghiniana by spectrally-resolved picosecond fluorescence at 77 K. Biochimica et Biophysica Acta-Bioenergetics. 1837 (6), 899-907 (2014).
- Ghazaryan, A., Akhtar, P., Garab, G., Lambrev, P. H., Büchel, C. Involvement of the Lhcx protein Fcp6 of the diatom Cyclotella meneghiniana in the macro-organisation and structural flexibility of thylakoid membranes. Biochimica Et Biophysica Acta-Bioenergetics. 1857 (9), 1373-1379 (2016).
- Darley, W. M. Biochemical composition. The biology of diatoms. 13, 198-223 (1977).
- Milsman, M. H. W., Schwendner, R. A., Weder, H. G. Preparation of large single bilayer liposomes by a fast and controlled dialysis. Biochimica Et Biophysica Acta. 512 (1), 147-155 (1978).
- Zumbuehl, O., Weder, H. G. Liposomes of controllable size in the range of 40 to 180 nm by defined dialysis of lipid-detergent-mixed micelles. Biochimica Et Biophysica Acta. 640 (1), 252-262 (1981).
- Verchere, A., Broutin, I., Picard, M. Photo-induced proton gradients for the in vitro investigation of bacterial efflux pumps. Scientific Reports. 2 (306), (2012).
- Chevallet, M., Luche, S., Rabilloud, T. Silver staining of proteins in polyacrylamide gels. Nature Protocols. 1 (4), 1852-1858 (2006).
