Name: native cell membrane nanoparticles system for membrane protein-protein interaction analysis
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这项研究得到了VCU创业基金（Y.G.）和国家卫生研究院国家普通医学研究所的支持，该奖项编号为R01GM132329（Y.G.）内容完全由作者负责，不一定代表国家卫生研究院的官方意见。我们感谢蒙特塞拉特·萨姆索和凯文·麦克罗伯茨对录像的慷慨支持。

1. 蛋白质表达
<ol><li>用BL21（DE3）plysS细胞特异性质粒的抗生素接种15 mL的"了不起的肉汤"（TB）介质，在37°C的50mL管中过夜表达质粒，在250 rpm时摇晃。</li>
	<li>检查 600 nm （OD 600） 的隔夜文化的光学密度，并确保其超过 2.0。</li>
	<li>将5毫升的细胞培养物稀释成1升含有质粒特异性抗生素的结核病介质，并在37°C下孵育，摇晃至OD 600=0.8，然后诱导最终浓度为1mM的IPTG。</li>
	<li>在20°C下...

<ol>
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(90), e51087 (2014).</li><li>Lee, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+detergent-free+isolation+of+membrane+proteins+in+their+local+lipid+environment.">A method for detergent-free isolation of membrane proteins in their local lipid environment.</a> Nature Protocols. 11 (7), 1149-1162 (2016).</li><li>Hall, S. C. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+acid-compatible+co-polymer+for+the+solubilization+of+membranes+and+proteins+into+lipid+bilayer-containing+nanoparticles.">An acid-compatible co-polymer for the solubilization of membranes and proteins into lipid bilayer-containing nanoparticles.</a> Nanoscale. 10 (22), 10609-10619 (2018).</li><li>Oluwole, A. 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蛋白质与蛋白质的相互作用对于膜蛋白结构和功能的完整性非常重要。已经开发出许多方法来研究蛋白质与蛋白质的相互作用。与可溶性蛋白相比，膜蛋白及其PPI由于膜蛋白的独特内在特性而更难研究。这一困难主要来自于要求将膜蛋白嵌入原生脂质双层环境中，以获得结构稳定性和功能性。这成为问题，因为为了确定蛋白质的高分辨率结构，他们必须从细胞膜中提取。FRET是研究细胞膜上蛋白质...

蛋白质-蛋白质相互作用（PPI）在整个生物学中起着关键作用，从蛋白质的结构和功能的维持到整个系统的调节。PPI 有多种不同的形式，可以根据它们形成的交互类型进行分类。其中一种分类是同质的还是异质的，基于是相同的亚单位之间的相互作用，还是作为亚单位的不同蛋白质之间的相互作用。另一个分类基于交互强度，如果相互作用导致稳定复合体或瞬态复合状态的形成。蛋白质之间PPI的结构信息对于理解蛋白质发挥其功能的机制至关重要。据估计，超过80%的蛋白质依靠复杂的形成来发挥其功能作用。鉴于观察到依赖PPI进行适当与生俱来的蛋白质在生物学中的重要性的百分比是显而易见的，但是能够正确研究参与这些相互作用的蛋白质仍然具有挑战性，因为可用于实验观察蛋白质何时形成PPI的技术存在限制。
由于目前许多可用的PPI确定技术产生的噪音、误报和假阴性，许多实验确定的PPI的结果存在高度的差异。对于酵母双混合（Y2H）系统、串联亲和力纯度-质谱（TAP-MS）和荧光共振能量转移（FRET）尤其如此，它们代表了PPI测定2、3、4、5、6、7三种最常用的方法。相比之下，蛋白质结构生物学技术，如核磁共振（NMR）、X射线晶体学和电子显微镜（EM），可用于获取有关蛋白质-蛋白质相互作用的高分辨率结构信息，直至原子水平，并允许直接直观地确认感兴趣的目标蛋白质发生的相互作用。所有目前可用的基于结构的PPI确定研究技术（例如Y2H、TAP-MS和FRET）都缺乏这种能力，此外还难以识别蛋白质5之间的弱和瞬态相互作用。这些缺点在研究膜蛋白时进一步增强，因为脂质环境的额外变量增加了复杂性，影响了适当的第四纪结构和异构复合组装的形成。
膜蛋白占蛋白质组的很大一部分，已知在所有生物体内的正常细胞功能中起着许多关键作用。尽管膜蛋白估计占人类基因组的27%，占目前所有药物靶点的60%，但膜蛋白的已解模型数量存在重大异常，仅占已公布的所有蛋白质结构8、9、10的2.2%。结构信息可用性差异的主要原因是膜蛋白本身的内在特性。由于其溶解性差，依赖与脂质环境的相互作用来维持原生结构和功能，以及脂膜本身的各种物理化学特性，膜蛋白在实施结构生物学技术研究时继续是一个实质性问题。在这些内在属性中，获取准确的结构信息最重要的是要求它们与其自然脂质环境相互作用，以保持其原生结构和功能。脂质环境是膜蛋白结构和功能不可分割的一部分，因此，膜素的概念（膜和蛋白质的组合）被提出为膜蛋白结构和功能的基本单位11。尽管脂蛋白相互作用很重要，但目前可用的基于结构的PPI测定技术往往要求所研究的蛋白质是可溶性的或用洗涤剂溶解的。暴露在苛刻的洗涤剂中会使蛋白质变质，或由于熟食而引起误报和阴性，从而诱发蛋白质的聚集、蛋白质的自然、非共价相互作用的分离以及人工寡头状态的形成。由于有必要保持一个自然脂质环境，以准确确定膜蛋白的原生寡聚状态，我们开发了一个原生细胞膜纳米粒子系统（NCMN）12基于先前报道的苯乙烯马累酸脂质颗粒（SMALP）13方法。
SMALP使用苯乙烯雄性酸共聚物作为膜活性聚合物提取和溶解膜蛋白。聚（苯乙烯共马力酸）（SMA）是一种独特的两栖聚合物，因为它的疏水苯乙烯潮湿和截然相反的亲水性马力酸莫伊蒂。它通过吸收、破坏稳定和破坏pH依赖机制14中的细胞膜来形成纳米粒子。SMA的这种功能活性使得它能够被用作膜蛋白提取和溶解的无洗涤剂系统。NCMN 系统在几个方面与 SMALP 系统不同。NCMN系统最独特的特点是，它有一个膜活性聚合物库，具有多种聚合物，具有独特的特性，使它们适合分离许多不同的膜蛋白，需要不同的条件，以稳定性和原生功能。与SMALP方法相比，NCMN系统在制备纳米粒子方面也有不同的协议。其中一个例子是，NCMN使用单步镍亲和力柱纯化进行高分辨率结构测定：在比较产生 3.2 和 3.0 é 低温 EM AcrB 结构的 NCMN 协议时，可以观察到这些不同协议的效果，并使用 SMALP 方法进行类似的研究，结果在 8.8 分辨率12、15分辨率下形成了低温 EM AcrB 结构。NCMN 系统的这些独特功能是以前建立的 SMALP 方法的改进，使其成为 PPI 研究的理想候选者。
多药排泄物运输器AcrB在大肠杆菌12中作为功能性同质体存在。诱变分析表明，单个 P223G 点突变位于负责 AcrB 修剪器稳定的环上，破坏修剪器状态的稳定性，并在用洗涤剂 DDM 制备时产生 AcrB 单体，可使用原生蓝胶电泳16检测。然而，FRET对AcrB-P223G突变体的分析表明，当在原生细胞膜脂质双层环境中，大多数突变的AcrB-P223G仍以修剪器形式存在，野生型AcrB和AcrB-P223G的表达水平相似。然而，尽管FRET的分析结果，对突变运输机的活动分析表明，与野生16型相比，活动急剧减少。虽然FRET技术已被普遍用于蛋白质-蛋白质相互作用的分析，但研究表明，它经常会产生误报17、18、19、20。
最近，通过使用NCMN12，确定了AcrB修剪器的高分辨率低温-EM结构，这表明AcrB与其相关的原生细胞膜脂双层的相互作用。原则上，NCMN可以很容易地用于分析在原生细胞膜上发现的蛋白质-蛋白质相互作用。在该系统的测试中，利用原生细胞膜纳米粒子和负染色电子显微镜进行实验，直接观察AcrB-P223G的寡头状态。为了与野生型AcrB纳米粒子进行比较，我们使用了相同的膜活性聚合物（SMA2000在我们的实验室中制造，在NCMN库中索引为NCMNP1-1），用于高分辨率低温-EM结构测定AcrB和NCMN库12中发现的替代聚合物。根据先前报告的结果，预计大多数AcrB-P223G突变体将以三元原生细胞膜纳米粒子21的形式存在。然而，在样品中未发现 AcrB 修剪器，例如与野生型 AcrB 一起观察到的修剪器。这表明大多数AcrB-P223G不会像先前建议的那样在原生细胞膜上形成修剪器。
在这里，我们报告与使用 NCMN 的野生大肠杆菌 AcrB 型相比，对突变大肠杆菌 AcrB-P223G 进行了详细分析。AcrB的这一案例研究表明，NCMN是蛋白质-蛋白质相互作用分析的良好系统。

<table>
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>30% Acrylamide/BIS SOL (37.5:1)</td>
			<td>Bio-Rad</td>
			<td>161-0158</td>
			<td></td>
		</tr>
		<tr>
			<td>4x Laemmli Sample Buffer (Loading Buffer)</td>
			<td>Bio-Rad</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic Acid Glacial</td>
			<td>ThermoFisher Scientific</td>
			<td>A38S-212</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium Persulfate (APS)</td>
			<td>Bio-Rad</td>
			<td>161-0700</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Goldbio</td>
			<td>C-105-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Coomassie Brilliant Blue R-250 protein stain powder</td>
			<td>Bio-Rad</td>
			<td>161-0400</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT (Dithiothreitol) (&#62; 99% pure) Protease free</td>
			<td>Goldbio</td>
			<td>DTT10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>ThermoFisher Scientific</td>
			<td>G33-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>ThermoFisher Scientific</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Affymetrix</td>
			<td>17525 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Goldbio</td>
			<td>I2481C100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Goldbio</td>
			<td>K-120-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate</td>
			<td>ThermoFisher Scientific</td>
			<td>AA3622636</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>ThermoFisher Scientific</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylethylenediamine (EDTA)</td>
			<td>Merck</td>
			<td>8.03779.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>NCMNS-P5-2</td>
			<td>Not commercially available yet</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>SDS (Sodium Dodecyl Sulfate)</td>
			<td>Bio-Rad</td>
			<td>161-0301</td>
			<td></td>
		</tr>
		<tr>
			<td>SMA2000</td>
			<td>Cray Valley</td>
			<td>Submit request for obtaining to corresponding author</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>S271-10</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP-HCl</td>
			<td>Goldbio</td>
			<td>TCEP25</td>
			<td></td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Bio-Rad</td>
			<td>161-0800</td>
			<td></td>
		</tr>
		<tr>
			<td>Terrific Broth Media</td>
			<td>Affymetrix</td>
			<td>75856 1 KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base</td>
			<td>Bio-Rad</td>
			<td>161-0719</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl Acetate</td>
			<td>Ambinter</td>
			<td>Amb22348393</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti J-26S XPI</td>
			<td>Beckman Coulter</td>
			<td>B14538</td>
			<td></td>
		</tr>
		<tr>
			<td>Avanti JXN-30</td>
			<td>Beckman Coulter</td>
			<td>B34193</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbon Electron Microscope Grids (10 nm)</td>
			<td>Electron Microscopy Sciences</td>
			<td>CF300-Cu-TH</td>
			<td></td>
		</tr>
		<tr>
			<td>Con-Torque Tissue Homogenizer</td>
			<td>Eberbach</td>
			<td>E7265</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning LSE Mini Microcentrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>07-203-954</td>
			<td></td>
		</tr>
		<tr>
			<td>EmulsiFlex-C3</td>
			<td>Avestin</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fraction Collector F9-R</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29003875</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-PROTEAN Tetra Vertical Electrophoresis Cell</td>
			<td>Bio-Rad</td>
			<td>165-8004</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>ThermoFisher Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr>
			<td>Optima L-90K Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>PN LL-IM-12AB</td>
			<td></td>
		</tr>
		<tr>
			<td>PELCO easiGlow Glow Discharge Cleaning System</td>
			<td>Ted Pella</td>
			<td>91000S-230</td>
			<td></td>
		</tr>
		<tr>
			<td>Potter-Elvehjem Safe Grind Tissue Grinder</td>
			<td>Wheaton</td>
			<td>358013</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr>
			<td>Razel R99-E Variable Speed Syringe Pump</td>
			<td>Razel Scientific Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200 Increase 10/300 GL</td>
			<td>GE Healthcare Life Sciences</td>
			<td>28990944</td>
			<td></td>
		</tr>
		<tr>
			<td>Tecnai F20 200kV</td>
			<td>FEI</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Type 70 Ti Fixed-Angle Rotor</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>General Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 ml Microcentrifuge Tubes</td>
			<td>ThermoFisher Scientific</td>
			<td>05-408-129</td>
			<td></td>
		</tr>
		<tr>
			<td>4 ml Amicon Ultra-4 30 kDa</td>
			<td>Millipore Sigma</td>
			<td>UFC803024</td>
			<td></td>
		</tr>
		<tr>
			<td>AKTA pure 25 L1 FPLC</td>
			<td>GE Healthcare Life Sciences</td>
			<td>29018225</td>
			<td></td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS Cells</td>
			<td>ThermoFisher Scientific</td>
			<td>C606003</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon 50 ml Conical Centrifuge Tube</td>
			<td>ThermoFisher Scientific</td>
			<td>14-959-49A</td>
			<td></td>
		</tr>
		<tr>
			<td>HisTrap HP 5 ml Column</td>
			<td>GE Healthcare Life Sciences</td>
			<td>17524802</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-24a</td>
			<td>EMD Biosciences</td>
			<td>69749-3</td>
			<td></td>
		</tr>
	</tbody>
</table>

native cell membrane nanoparticles system for membrane protein-protein interaction analysis

细胞膜系统中的蛋白质-蛋白质相互作用在广泛的生物过程中起着至关重要的作用——从细胞到细胞相互作用到信号转导：从传感环境信号到生物反应;从代谢调节到发育控制。蛋白质-蛋白质相互作用的准确结构信息对于了解膜蛋白复合物的分子机制以及设计高特异性分子来调节这些蛋白质至关重要。已经开发出许多体内和体外方法，用于检测和分析蛋白质-蛋白质相互作用。其中结构生物学方法的独特之处在于，它可以在原子水平上提供蛋白质-蛋白质相互作用的直接结构信息。然而，目前的膜蛋白结构生物学仍然在很大程度上仅限于洗涤剂为基础的方法。基于洗涤剂的方法的主要缺点是，一旦洗涤剂分子去除其原生脂质双层环境，它们通常会分离或变性膜蛋白复合物。我们一直在开发一种用于膜蛋白结构生物学的原生细胞膜纳米粒子系统。在这里，我们演示了该系统在细胞膜上蛋白质-蛋白质相互作用的分析中的应用，以及AcrB的寡头状态的案例研究。

使用此处介绍的程序，纯化了 大肠杆菌 AcrB野生型和 大肠杆菌 突变AcrB-P223G的样品。然后，样品被吸附到碳负污渍电子显微镜网格和染色使用尿素醋酸盐与侧印方法22。使用传输电子显微镜收集负污渍图像。用DDM纯化的AcrB野生型样品的负污渍图像揭示了单分散颗粒的同质溶液，蛋白质显示一个定义明确的三角结构（图1A）。这些修剪结构与净?...

Watch this Scientific Journal Video about Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis at JoVE.com

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

这里介绍的是一个协议，用于确定膜蛋白的寡头状态，该方案利用本地细胞膜纳米粒子系统与电子显微镜配合使用。

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Research

JoVE Journal

Biochemistry

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

方法确定HCN1和TRIP8b之间蛋白 - 蛋白相互作用的小分子抑制剂

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

单分子水平的高精度FRET用于生物分子结构测定

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

蓝色本机凝胶电泳法分析囊体膜蛋白配合物

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

大肠杆菌中异构表达对膜迁移者的特性与膜囊泡的产生

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

热通菌膜-双磷酶抑制剂的筛选

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

重建线粒体膜动力学的模型膜平台

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial ...