单粒子冷冻电子显微镜在结构中测定脂质敏感分通道 trpc3 的表达与纯化
Summary
该协议描述了用低温电子显微镜测定离子通道结构的技术, 包括用于以最小的努力和毒性在哺乳动物细胞中有效表达基因的杆菌病毒系统, 蛋白质提取、纯化、和质量检查, 样品网格的制备和筛选, 以及数据的收集和处理。
Abstract
典型 trp 亚家族的瞬态受体电位通道 (trpc) 是非选择性阳离子通道, 在钙稳态中发挥着至关重要的作用, 特别是在商店操作的钙进入, 这对于保持钙的适当功能至关重要。突触囊泡释放和细胞内信号通路。因此, trpc 渠道与各种人类疾病有关, 包括心血管疾病, 如心肌肥大, 神经退行性疾病, 如帕金森病, 和神经疾病, 如脊柱小脑共济失调。因此, trpc 通道是人类疾病中潜在的药理靶点。然而, 在这些通道中门控的分子机制仍不清楚。在结构测定研究中, 特别是对于哺乳动物膜蛋白, 如 trpc 离子通道, 获得大量稳定、均匀和纯化蛋白的困难一直是一个限制因素。在这里, 我们提出了一个协议, 大规模表达哺乳动物离子通道膜蛋白使用一个修改后的巴库氏病毒基因转移系统和纯化这些蛋白质的亲和力和大小排除色谱。我们进一步提出了一个协议, 收集单粒子冷冻电子显微镜图像从纯化的蛋白质, 并使用这些图像来确定蛋白质的结构。结构确定是了解离子通道中门控和功能机制的有力方法。
Introduction
钙参与大多数细胞过程, 包括信号级联, 转录控制, 神经递质释放, 和激素分子合成1,2,3。细胞质游离钙的稳态维持对细胞的健康和功能至关重要。细胞内钙稳态的主要机制之一是储存钙进入 (sace), 在这个过程中, 储存在内质网 (er) 中的钙的耗尽会触发质膜上离子通道的打开, 以促进er 钙的补充, 然后可以在进一步信号4,5,6中使用。瞬态受体电位通道 (trpc) 是属于 trp 超家族的透钙通道, 已被确定为 soce7、8、9的主要参与者。
在 trpc 家族的7名成员中, trpc3、trpc6 和 trpc7 构成了同源亚群, 它们在被脂质二级信使二丙烯酸酯 (dag) 激活的能力上是独一无二的, 而二维信使二丙烯酸甘油是脂质信号的降解产物磷脂酰肌醇 4, 5-双磷酸 (pip2)10,11。trpc3 在平滑肌和大脑的大脑和小脑区域中高度表达, 在这些区域中, 它在影响神经传递和神经发生的钙信号中发挥着至关重要的作用。trpc3 功能障碍与中枢神经系统疾病、心血管疾病和某些癌症 (如卵巢腺癌14、15、16) 有关。因此, trpc3 有望成为治疗这些疾病的药物目标。由于对其分子激活机制, 包括脂质结合位点 17、18缺乏了解, 开发针对 trpc3 的有针对性的药物受到限制。我们报告了人类 trpc3 通道 (htrpc3) 及其两个处于封闭状态的脂质结合位点的第一个原子分辨率结构, 为这些机制提供了重要的见解19。
高分辨率膜蛋白结构的关键因素是获得高质量的蛋白质。相应的表达筛选和纯化条件的必要, 以获得高质量的蛋白质可能是一个耗时和昂贵的努力。在这里, 我们提出了一个协议, 详细描述了我们如何确定 htrpc3 的表达和纯化的最佳条件, 它在我们的初始筛选中表现不佳。我们就如何排除故障和优化蛋白质行为提出了几个要点, 为我们的低温电子显微镜 (低温 em) 研究奠定了坚实的基础。我们使用了由 gouaux 和他的同事开发的改良的杆菌病毒产生载体 (peg), 该载体经过优化, 用于在哺乳动物细胞20 中筛选检测和高效生成巴库索病毒。这种表达方法适用于哺乳动物细胞膜中蛋白质的快速和具有成本效益的过度表达。我们将此向量的使用与基于荧光检测大小排除色谱 (fsec) 预置方法21结合起来。该方法采用绿色荧光蛋白 (gfp) 标记融合到感兴趣的结构中, 并提高了目标蛋白在小的全细胞溶解性样品中的可视化。这样就可以在不同的洗涤剂和添加剂的存在下筛选蛋白质稳定性, 并具有稳定的热稳定性突变, 并允许使用少量细胞进行小规模瞬态转染。通过这种方式, 在进入大规模蛋白质纯化之前, 可以快速筛选多种条件。在表达、筛选和纯化之后, 我们提出了一种从低温 em 中获取和处理图像的协议, 以生成蛋白质的新结构测定。我们相信, 这里描述的方法将作为一个通用的协议, 用于 trp 通道受体和其他膜蛋白的结构研究。
Protocol
1. dh10α组成细胞转化为 bacm中 dna 合成感兴趣的基因, 并将其亚化为一个修改版本的 peg 向量, 其中包含一个双链球标记、一个 his8 标记和 gfp, 在 n 终点 (pfastbaci) 20 处有凝血酶裂解部位。 通过在 1.5 ml 管中加入5纳克含有所需基因的质粒, 在 pFastBacI 中加入50μl 的 dh10α细胞, 并在冰上孵育 10分钟, 从而转化合格的细胞。在42°c 下, 热冲击细胞45秒。在管中加入200μl 的超最…
Representative Results
图 1a 显示了 htrpc3 表达和纯化协议的示意图。图 1b 显示了 htrpc3 带理想白色菌落的图像, 类似于为 bacmacmt dna 纯化选择的基板。我们发现, 48小时是理想的明确蓝甲染色, 同时保持孤立的殖民地的存在。Sf9 荧光显示, htrpc3 p2 病毒的峰值产量是在 sf9 昆虫细胞感染 4 d 后出现的 (图 1c)。 <p class="jove_content" fo:ke…
Discussion
在过去几年里, 由于开发了新的摄像机和算法, 显著加快了蛋白质的结构测定, 而这些相机和算法并没有显著加快蛋白质的结构测定, 因此, 用低温 em 对蛋白质进行结构测定已经给结构生物学领域带来了革命性的变化。容易结晶, 特别是膜蛋白。尽管冷冻-em 技术最近取得了所有进展, 但在质量和数量上足以促进高质量成像的纯化蛋白的制备通常仍然是耗时、昂贵和具有挑战性的。如上述协议所述, 能?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
我们感谢赵先生和孟先生在大卫·范安德尔高级冷冻电子显微镜套房的数据收集方面给予的支持。我们感谢 vari 高性能计算团队提供的计算支持。我们感谢 n. clemente、d. dues、j. floramo、y. 黄、y. kim、c. mueller、b. roth 和 z. ruan 提出的意见, 这些评论大大改进了这份手稿。我们感谢 d. nadziejka 对这份手稿的编辑支持。这项工作得到了 vari 内部资金的支持。
Materials
| pEG BacMam vector (pFastBacI) | addgene | 31488 | |
| DH10α cells | Life Technologies | 10361-012 | |
| S.O.C. media | Corning | 46003CR | for transformation of DH10α cells for Bacmid |
| Bacmam culture plates | Teknova | L5919 | for culture of transformed DH10α cells |
| Incubation shaker for bacterial cells | Infors HT | Multitron standard | |
| Incubated orbital shaker for insect cells | Thermo-Fisher | SHKE8000 | |
| Reach-in CO2 incubator for mammalian cells | Thermo-Fisher | 3951 | |
| Table-top orbital shaker | Thermo-Fisher | SHKE416HP | used in Reach-in CO2 incubator for mammalian cells |
| Incubator | VWR | 1535 | for bacterial plates |
| QIAprep Spin Miniprep Kit | Qiagen | 27106 | for plasmid extraction and purification |
| Phenol:Chloroform:Isoamyl alcohol | Invitrogen | 15593031 | for DNA extraction |
| Sf9 cells | Life Technologies | 12659017 | insect cells for producing virus |
| Sf-900 media | Gibco | 12658-027 | insect cell media |
| FBS | Atlanta Biologicals | S11550 | |
| Cellfectin II | Gibco | 10362100 | for transfecting insect cells |
| lipofectamine 2000 | Invitrogen | 11668-027 | for transfecting mamalian cells |
| 0.2 mm syringe filter | VWR | 28145-501 | for filtering P1 virus |
| 0.2 mm filter flasks 500ml resevoir | Corning | 430758 | for filtering P2 virus |
| erlenmeyer culture flask (flat bottom 2L) | Gene Mate | F-5909-2000 | for culturing insect cells |
| erlenmeyer culture flask (baffled 2L) | Gene Mate | F-5909-2000B | for culturing mammalian cells |
| nanodrop 2000 spectrophotometer | Thermo-Fisher | ND-2000 | for determining DNA and protein concentrations |
| HEK293 | ATCC | CRL-3022 | mammalian cells for producing protein |
| Freestyle 293 expression Medium | Gibco | 1238-018 | mammalian cell media for protein expression |
| Butyric Acid Sodium Salt | Acros | 263195000 | to amplify protein expression |
| PMSF | Acros | 215740500 | protease inhibitor |
| Aprotinin from bovine lung | Sigma-Aldrich | A1153-100MG | protease inhibitor |
| Leupeptin hydrochloride | Sigma-Aldrich | 24125-16-4 | protease inhibitor |
| pepstatin A | Fisher Scientific | BP2671-250 | protease inhibitor |
| digitonin | EMD Millipore | 300410 | detergent – to solubilize protein from membrane |
| imidazole | Sigma | 792527 | to elute protein from resin column |
| TALON resin | Clonetech | 635504 | for affinity purification by His-tag |
| superose6 incease columns | GE | 29091596; 29091597 | for HPLC and FPLC |
| Prominence Modular HPLC System | Shimadzu | See Below | |
| Controller Module | " | CBM20A | |
| Solvent Delivery System | " | LC30AD | |
| Fluorescence Detector | " | RF20AXS | |
| Autosampler with Cooling | " | SIL20ACHT | |
| Pure FPLC System with Fractionator | Akta | ||
| thrombin (alpha) | Haematologic Technologies Incorporated | HCT-0020 Human alpha | for cleaving GFP tag |
| Amicon Ultra 15 mL 100K centrifugal filter tube | Millipore | UFC910008 | for concentrating protein |
| EDTA | Fisher | E478500 | for stabilizing protein |
| 400 mesh carbon-coated copper grids | Ted Pella Inc. | 01754-F | grids for negative stain |
| Quantifoil holey carbon grid (gold, 1.2/1.3 μm size/hole space, 300 mesh) | Electron Microscopy Sciences | Q3100AR1.3 | grids for Cryo-EM |
| Vitrobot Mark III | FEI | for preparing sample grids by liquid ethane freezing | |
| liquid nitrogen | Dura-Cyl | UN1977 | |
| ethane gas | Airgas | UN1035 | |
| Solarus Plasma System | Gatan | Model 950 | for cleaning grids before sample freezing |
| Tecnai Spirit electron microscope | FEI | for negative stain EM imaging | |
| Talos Arctica electron microsocope | FEI | for screening and low resolution imaging of Cryo-EM grids | |
| Titan Krios electron microscope | FEI | for high-resolution Cryo-EM imaging | |
| Software | |||
| Gautomatch software | http://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ | to pick particles from micrographs | |
| Relion 2.1 software | https://github.com/3dem/relion | to construct 2D and 3D classification | |
| CryoSPARC software | https://cryosparc.com/ | to generate an initial structure model | |
| Frealign software | http://grigoriefflab.janelia.org/frealign | to refine particles | |
| Coot software | https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | to build a model | |
| MolProbity software | http://molprobity.biochem.duke.edu/ | to evaluate the geometries of the atomic model | |
| SerialEM software | http://bio3d.colorado.edu/SerialEM/ | for automated serial image stack acquisition | |
| MortionCor2 software | http://msg.ucsf.edu/em/software/motioncor2.html | for motion correction of summed movie stacks | |
| GCTF software | https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/ | for measuring defocus values in movie stacks | |
| Phenix.real_space_refine software | https://www.phenix-online.org/documentation/reference/real_space_refine.html | for real space refinement of the initial 3D model |
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Citar este artículo
Haley, E., Choi, W., Fan, C., Sun, W., Du, J., Lü, W. Expression and Purification of the Human Lipid-sensitive Cation Channel TRPC3 for Structural Determination by Single-particle Cryo-electron Microscopy. J. Vis. Exp. (143), e58754, doi:10.3791/58754 (2019).