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Bioquímica

使用脂质单层方法进行电子晶体学研究的样品制备

Published: November 20, 2021
doi:
10.3791/63015
, , , ,
1School of Molecular Sciences,Arizona State University, 2Center for Applied Structural Discovery, Biodesign Institute,Arizona State University, 3Eyring Materials Center,Arizona State University, 4Department of Microbiology and Immunology,The Pennsylvania State University

Summary

几十年来，脂质单层一直被用作形成二维（2D）蛋白质晶体的基础，用于结构研究。它们在气水界面上稳定，可作为电子成像的薄支撑材料。在这里，我们介绍了为生物研究准备脂质单层的行之有效的步骤。

Abstract

电子晶体学是高分辨率结构测定的有力工具。可溶性或膜蛋白等大分子可以在有利条件下生长成高有序的二维（2D）晶体。已种植的 2D 晶体的质量对于通过 2D 图像处理最终重建的分辨率至关重要。多年来，脂质单层已被用作支撑层，以培养外周膜蛋白和可溶性蛋白的二元结晶。该方法还可用于整体膜蛋白的二元结晶，但需要更广泛的经验调查，以确定洗涤剂和透析条件，以促进分隔到单层。在空气-水接口形成脂质单层，使极地脂质头组在水分相中保持水分，非极性乙酰链条、尾部分裂到空气中，打破表面张力，使水面变平。头部组的带电性质或独特的化学形态为溶液中的蛋白质提供了亲和力，促进二维阵列形成的结合。带有 2D 阵列的新形成的单层器可以很容易地转移到碳涂层铜网格上的电子显微镜 （EM）中，用于提升和支持晶体阵列。在这项研究中，我们描述了低温电子微观（低温-EM）成像的脂质单层方法。

Introduction

通过2D晶体或蛋白质螺旋阵列的电子衍射，在有利的情况下可以达到亚纳米分辨率1，2，3。特别感兴趣的是重建的二元膜蛋白质阵列或晶体在其近本土环境1。由于晶体充当信号放大器，可增强特定空间频率下结构因子的强度，因此电子晶体学允许探测比单粒子低温-EM尺寸较小的目标，如小分子。电子束可以通过有序的二维蛋白质阵列衍射，生成衍射模式或晶格图像，具体取决于图像平面记录在探测器4上的位置。然后，可以提取和处理衍射强度，以重建晶体的二维投影结构。电子的散射横截面比X射线大，其散射主要遵循卢瑟福模型，该模型基于电子与分子5中带电原子之间的库隆布相互作用。2D膜晶体的厚度通常小于100纳米，适合在标本6内不发生动态散射的电子传输。电子晶体学研究已被证明是探测膜蛋白和脂蛋白相互作用7、8、9、10、11、12、13、14、15、16、17的高分辨率结构信息的有力工具。

脂质单层是一个单脂层，由空气-水接口6密集包装的磷脂组成，可协助可溶性蛋白质或外周膜蛋白18的二元阵列形成。根据脂质的密度及其横向压力，脂质分子可以在空气-水接口上形成有序的二维阵列，其乙酰链延伸到空气中，亲水性头组暴露在水溶液1，6，19。脂质头组可以通过静电相互作用与蛋白质相互作用，也可以进行修改，以提供亲和力标记来结合特定的蛋白质域。例如， DOGS-NTA-Ni （1，2-二恶英-sn-甘油-3-[（N-（5-氨基-1-卡盒式）氨基酸）苏奇尼）2-尼2+）常用于形成脂质单层，将蛋白质与多组蛋白标签20，21，22结合。此外，霍乱毒素B可以结合一个特殊的五氯苯甲酸GM1在脂质单层结构研究23，24。通过将蛋白质固定在脂质头组上，脂质单层可以协助形成用于高分辨率电子晶体学研究的薄的 2D 阵列。脂质单层技术已用于蛋白质结构研究的电子晶体学， 如链球菌素2，25，附件素V26，霍乱毒素27，大肠杆菌B亚单位28，大肠杆菌RNA聚合酶25，29，30，卡盒体壳蛋白31和HIV-132和莫洛尼穆林白血病病毒的辣椒蛋白33.由于脂质单层的稳定性和化学特性，已探索了冷冻-EM成像34的样品制备应用。但是，蛋白质阵列的形成需要优化。

在这里，我们提供了关于脂质单层器用于低温-EM成像的一般准备的广泛细节，以及一些可能影响已形成单层质量的考虑。

Protocol

1. 特氟隆块准备 从耐化学PTFE（聚氟乙烯）树脂中制备特氟龙块。使用一般钻头在块上打洞，然后用 图1中标出的尺寸。 2. 单层脂质制剂 注：估计运行时间：30-45分钟 脂质库存准备 使用 8.91 mL 氯仿/甲醇、0.99 mL 甲醇和 0.01 mL 10 毫克/mL 脂质，在 9：1 （v/v） 氯仿/甲醇中准备 0.01 毫克/…

Representative Results

沉积在 EM 网格上的脂质单层可以在传输电子显微镜 （TEM） 下可视化，而不会弄脏。单层存在可以通过与光束路径中没有任何标本的区域对比差来识别。与无覆盖区域相比，脂质单层覆盖区域的局部对比度较低，因为穿过空孔的电子束没有散射，显示更亮的照明（图3）。 要筛选 2D 单层结晶的条件，使用负污渍 EM 或低温 EM 成像调查标本的图像。当舞台?…

Discussion

脂质单层是促进大型二元晶体生长的强大工具，用于生物大分子的结构研究。为了在空气-水接口成功准备一个完整的脂质单层，强烈建议在实验当天新鲜制备脂质，因为脂质乙酰链的氧化可能导致单层包装中断，并对由此产生的晶体形成产生不利影响。购买的粉末形式的脂质应使用氯仿的混合物溶解：甲醇溶剂，这是通常用来溶解磷脂35。氯仿：甲醇溶剂可以预制并储存在密封?…

Divulgaciones

The authors have nothing to disclose.

Acknowledgements

这份手稿的编写得到了美国陆军研究办公室（W911NF2010321）和亚利桑那州立大学启动基金对P.-L.C的部分支持。

Materials

14:0 PC (DMPC) Avanti Lipids 850345 1,2-dimyristoyl-sn-glycero-3-phosphocholine,
1 x 25 mg, 10 mg/mL, 2.5 mL
Bulb for small pipets Fisher Scientific 03-448-21
Chloroform Sigma-Aldrich C2432
Desiccator vacuum Southern Labware 55207
EM grids Electron Microscopy Sciences CF413-50 CF-1.2/1.3-4C 1.2 µm hole, 1.3 µm space
Filter paper GE Healthcare Life Sciences 1001-090 Diameter 90 mm
Glass Pasteur pipets Fisher Scientific 13-678-20A
Hamilton syringe (25 µL) Hamilton Company 80465
Hamilton syringe (250 µL) Hamilton Company 81165
Hamilton syringe (5 µL) Hamilton Company 87930
Hamilton syringe (500 µL) Hamilton Company 203080
Methanol Sigma-Aldrich M1775-1GA
Petri dish VWR 25384-342 100 mm × 15 mm
Teflon block Grainger 55UK05 60 µL wells with side injection ports, manually made
Tweezers Electron Microscopy Sciences 78325 Various styles
Ultra-pure water
Ultrasonic cleaner VWR 97043-996
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Referencias

  1. Raunser, S., Walz, T. Electron crystallography as a technique to study the structure on membrane proteins in a lipidic environment. Annual Review of Biophysics. 38 (1), 89-105 (2009).
  2. Avila-Sakar, A. J., Chiu, W. Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography. Biophysical Journal. 70 (1), 57-68 (1996).
  3. Braun, T., Engel, A. Two-dimensional electron crystallography. Nature Encyclopedia of Life Sciences. , (2004).
  4. Wang, H. -. W., Wang, J. -. W. How cryo-electron microscopy and X-ray crystallography complement each other. Protein Science: a publication of the Protein Society. 26 (1), 32-39 (2017).
  5. Williams, D. B., Carter, C. B. . Transmission electron microscopy. , (2016).
  6. Abeyrathne, P. D., et al. 1.15 Analysis of 2-D Crystals of Membrane Proteins by Electron Microscopy. Comprehensive Biophysics. , 277-310 (2012).
  7. Muller, M. P., et al. Characterization of Lipid-Protein Interactions and Lipid-Mediated Modulation of Membrane Protein Function through Molecular Simulation. Chemical Reviews. 119 (9), 6086-6161 (2019).
  8. Martínez-Ballesta, M. D. C., Carvajal, M. Mutual Interactions between Aquaporins and Membrane Components. Frontiers in Plant Science. 7, 1322 (2016).
  9. Hite, R. K., Chiu, P. -. L., Schuller, J. M., Walz, T. Effect of lipid head groups on double-layered two-dimensional crystals formed by aquaporin-0. PloS One. 10 (1), 0117371 (2015).
  10. Murata, K., et al. Structural determinants of water permeation through aquaporin-1. Nature. 407 (6804), 599-605 (2000).
  11. Schenk, A. D., et al. The 4.5 A structure of human AQP2. Journal of Molecular Biology. 350 (2), 278-289 (2005).
  12. Gonen, T., et al. Lipid-protein interactions in double-layered two-dimensional AQP0 crystals. Nature. 438 (7068), 633-638 (2005).
  13. Hiroaki, Y., et al. Implications of the aquaporin-4 structure on array formation and cell adhesion. Journal of Molecular Biology. 355 (4), 628-639 (2006).
  14. Gonen, T., Sliz, P., Kistler, J., Cheng, Y., Walz, T. Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature. 429 (6988), 193-197 (2004).
  15. Chiu, P. -. L., et al. The structure of the prokaryotic cyclic nucleotide-modulated potassium channel MloK1 at 16 A resolution. Structure. 15 (9), 1053-1064 (2007).
  16. Kowal, J., et al. Ligand-induced structural changes in the cyclic nucleotide-modulated potassium channel MloK1. Nature Communications. 5, 3106 (2014).
  17. Walz, T., Grigorieff, N. Electron Crystallography of Two-Dimensional Crystals of Membrane Proteins. Journal of Structural Biology. 121 (2), 142-161 (1998).
  18. Yeager, M., Dryden, K. A., Ganser-Pornillos, B. K. Lipid monolayer and sparse matrix screening for growing two-dimensional crystals for electron crystallography: methods and examples. Methods in Molecular Biology. 955, 527-537 (2013).
  19. Pal, S. Chapter 6 – Structure analysis and visualization. Fundamentals of Molecular Structural Biology. , 119-147 (2020).
  20. Frey, W., et al. Two-dimensional protein crystallization via metal-ion coordination by naturally occurring surface histidines. Proceedings of the National Academy of Sciences of the United States of America. 93 (10), 4937-4941 (1996).
  21. Kubalek, E. W., Le Grice, S. F., Brown, P. O. Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid. Journal of Structural Biology. 113 (2), 117-123 (1994).
  22. Vénien-Bryan, C., et al. Structural study of the response regulator HupR from Rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid. Journal of Molecular Biology. 274 (5), 687-692 (1997).
  23. Merritt, E. A., Sarfaty, S., vanden Akker, F., L’Hoir, C., Martial, J. A., Hol, W. G. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Science: a publication of the Protein Society. 3 (2), 166-175 (1994).
  24. Mosser, G., Mallouh, V., Brisson, A. A 9 A two-dimensional projected structure of cholera toxin B-subunit-GM1 complexes determined by electron crystallography. Journal of Molecular Biology. 226 (1), 23-28 (1992).
  25. Edwards, A. M., Darst, S. A., Hemming, S. A., Li, Y., Kornberg, R. D. Epitaxial growth of protein crystals on lipid layers. Nature Structural Biology. 1 (3), 195-197 (1994).
  26. Olofsson, A., Mallouh, V., Brisson, A. Two-dimensional structure of membrane-bound annexin V at 8 A resolution. Journal of Structural Biology. 113 (3), 199-205 (1994).
  27. Ribi, H. O., Ludwig, D. S., Mercer, K. L., Schoolnik, G. K., Kornberg, R. D. Three-dimensional structure of cholera toxin penetrating a lipid membrane. Science. 239 (4845), 1272-1276 (1988).
  28. Celia, H., et al. Three-dimensional model of Escherichia coli gyrase B subunit crystallized in two-dimensions on novobiocin-linked phospholipid films. Journal of Molecular Biology. 236 (2), 618-628 (1994).
  29. Darst, S. A., Kubalek, E. W., Kornberg, R. D. Three-dimensional structure of Escherichia coli RNA polymerase holoenzyme determined by electron crystallography. Nature. 340 (6236), 730-732 (1989).
  30. Schultz, P., et al. Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals. Journal of Molecular Biology. 216 (2), 353-362 (1990).
  31. Dryden, K. A., Crowley, C. S., Tanaka, S., Yeates, T. O., Yeager, M. Two-dimensional crystals of carboxysome shell proteins recapitulate the hexagonal packing of three-dimensional crystals. Protein Science: a publication of the Protein Society. 18 (12), 2629-2635 (2009).
  32. Barklis, E., McDermott, J., Wilkens, S., Fuller, S., Thompson, D. Organization of HIV-1 capsid proteins on a lipid monolayer. The Journal of BIOLOGICAL CHemistry. 273 (13), 7177-7180 (1998).
  33. Barklis, E., et al. Structural analysis of membrane-bound retrovirus capsid proteins. The EMBO Journal. 16 (6), 1199-1213 (1997).
  34. Kelly, D. F., Dukovski, D., Walz, T. Monolayer purification: a rapid method for isolating protein complexes for single-particle electron microscopy. Proceedings of the National Academy of Sciences of the United States of America. 105 (12), 4703-4708 (2008).
  35. Reis, A., Rudnitskaya, A., Blackburn, G. J., Mohd Fauzi, N., Pitt, A. R., Spickett, C. M. A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL. Journal of Lipid Research. 54 (7), 1812-1824 (2013).
  36. Ueda, E. K. M., Gout, P. W., Morganti, L. Current and prospective applications of metal ion-protein binding. Journal of Chromatography. A. 988 (1), 1-23 (2003).
  37. Dietrich, J., nien-Bryan, C. . Strategies for Two-dimensional Crystallization of Proteins Using Lipid Monolayers. , (2005).
  38. Kuang, Q., Purhonen, P., Hebert, H. Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation. BioMed Research International. 2015, 693869 (2015).
  39. De Zorzi, R., Nicholson, W. V., Guigner, J. -. M., Erne-Brand, F., Vénien-Bryan, C. Growth of large and highly ordered 2D crystals of a K+ channel, structural role of lipidic environment. Biophysical Journal. 105 (2), 398-408 (2013).
  40. Johnson, M. C., Schmidt-Krey, I. Two-dimensional crystallization by dialysis for structural studies of membrane proteins by the cryo-EM method electron crystallography. Methods in Cell Biology. 113, 325-337 (2013).
  41. Rémigy, H. -. W., Caujolle-Bert, D., Suda, K., Schenk, A., Chami, M., Engel, A. Membrane protein reconstitution and crystallization by controlled dilution. FEBS Letters. 555 (1), 160-169 (2003).
  42. Braun, T., Kaufmann, T. C., Rémigy, H., Engel, A. Two-dimensional Crystallization of Membrane Proteins. Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. , 1936-1942 (2006).
  43. Lebeau, L., Vénien-Bryan, C. Monolayer two-dimensional crystallization of membrane proteins. Methods in Molecular Biology. 955, 59-71 (2013).
  44. Seddon, A. M., Curnow, P., Booth, P. J. Membrane proteins, lipids and detergents: not just a soap opera. Biochimica et Biophysica Acta. 1666 (1-2), 105-117 (2004).
  45. Lebeau, L., et al. Two-dimensional crystallization of a membrane protein on a detergent-resistant lipid monolayer. Journal of Molecular Biology. 308 (4), 639-647 (2001).

Tags

Lipid MonolayerElectron Crystallography2D CrystallizationMembrane ProteinsProtein BindingSample PreparationTeflon PlateBuffer ReservoirHumidity ControlCarbon-coated EM Grid

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Truong, C. D., Williams, D. R., Zhu, M., Wang, J. C., Chiu, P. Sample Preparation using a Lipid Monolayer Method for Electron Crystallographic Studies. J. Vis. Exp. (177), e63015, doi:10.3791/63015 (2021).
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