The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).

1.脚手架脂质体制剂注:在理想情况下，最初的实验应该使用一个相对简单的和特征的支架（包括DOPC（1,2- dioleoyl- SN -glycero -3-磷酸胆碱或PC）和DGS-NTA（镍2+）（1,2-二油酰基的- SN -glycero -3 - [（N - （5-氨基-1-羧基戊基）亚氨基二乙酸）琥珀酰基]（镍盐））建筑物关闭这些实验中，脂质电荷，柔韧性，和曲率可以引入作为单独因素同的电位，以改变膜近端相互作用。这些改变可以通过添加规定量的特定的脂质成分，包括磷脂酰丝氨酸或心磷脂（CL），磷脂酰乙醇胺（DOPE或PE），或半乳糖（β）神经酰胺来实现。 <ol><li>结合溶于氯仿在一个干净的玻璃试管脂质。同时旋转该管以形成薄脂质膜蒸发用干燥氮气除去溶剂。用centrifuga除去残留溶剂升蒸发器在37℃1小时。 注:各种脂质体制剂是在协议中使用如下所述:支架脂质体（3.3％（摩尔）DGS-NTA（镍离子 ）/ 96.7％摩尔DOPC），与心磷脂脂质体支架（3.3％（摩尔）DGS-NTA（镍离子 ） / 10％（摩尔）心磷脂/ 86.7％摩尔DOPC），灵活的支架与脂质体心磷脂（3.3％（摩尔）DGS-NTA（镍离子 ）/ 10％（摩尔）心磷脂/ 35％（摩尔）DOPE / 51.7％摩尔DOPC），以及丰富的脚手架脂质体（10％摩尔DGS-NTA（镍离子 ）/ 15％（摩尔）心磷脂/ 35％（摩尔）DOPE / 40％摩尔DOPC）。 </li><li>添加预热至37℃的缓冲液A（25mM的HEPES（4-（2-羟乙基）-1-哌嗪乙磺酸），150毫米氯化钾，pH值调节至7.5，用KOH），使得最终的脂质浓度为1 - 2毫米。孵育30分钟，在37℃偶尔涡旋充分悬浮的脂质混合物（ 图1a）。 </li><li>转移到塑料试管，将管在液氮中，直到完全冻结（roug溶血素30秒），并在37℃水浴直至完全解冻（约1处 - 2分钟）。重复，共4次冻融循环（ 图1b）。 </li><li>浸泡4滤波器支持和在缓冲液中的聚碳酸酯过滤器，并根据制造商的说明书组装挤出制备的脂质挤出机。通过过滤器21次挤出脂质溶液。使用温和，恒定压力，以确保均匀的尺寸分布（ 图1c）。 注:在本协议中所描述的所有实验中，用于挤出一个1.0微米的聚碳酸酯滤器。可与各种脂质体的直径为50至400nm 12或更大13的观察与阴离子脂质DRP1相互作用。因此，1微米的过滤器的尺寸选择为理想为GTP酶的活性和电子显微镜。如果其他脂质体直径的需要，在制备巨头单层脂质体14,15（GUVs）或小unilamell的芳囊泡16（越野车）都可以使用。动态光散射可以用于评估脂质体大小异质13。 </li><li>在4℃保存挤出脂质体和后3丢弃 - 5 D。 </li></ol> 2.蛋白结合分析使用脚手架的脂质体<ol><li>样品制备<ol><li>孵育His-标记MffΔTM（5μM终浓度）与骨架的脂质体（40％（摩尔）的PC / 35摩尔％的PE / 15摩尔％的CL / 10摩尔％的DGS-NTA（镍2+）;50μM的最终）为至少15分钟在RT缓冲液A + BME（25毫米的HEPES，150mM的氯化钾，10mM的β巯基乙醇（BME），pH值调节至7.5，用KOH）。对于MFF的自由控制，孵化用他的标记对照蛋白（如GFP）来绑定和盾暴露NTA（镍离子 ）的脂质体。 注:MffΔTM表达和纯化如在先前的研究2中所描述。绿色荧光蛋白以相似的方式进行纯化，但是省略了离子交换步骤。 BME被要求为这些experimenTS因为DRP1是氧化，其可以改变其活性和装配性能敏感。 </li><li>添加DRP1（2μM最终），并在室温下孵育1小时。 注:DRP1表达和纯化如在先前的研究2中所描述。与DRP1孵育后，核苷酸对膜变形结合的效果可通过温育一个附加小时了2mM的MgCl 2和任一1mM的GTP，1mM的GMP-PCP，或缓冲液A + BME进行调查。 </li></ol></li><li>负染色透射电子显微镜（EM）的分析<ol><li>转移5μL的样品的实验室膜的片，并放置在样品上的碳涂层Cu /铑网格。孵育样品上的栅极1分钟，吸干远在滤纸上多余的液体，并转移到2％乙酸双氧铀的下降。孵育1分钟，吸干滤纸上过量染色，并转移到网格框。在真空条件下O / N商店，以确保完全干燥。 </li><li>使用透射电子microsc图像样本OPE 18,500 - 30,000X放大观察的蛋白质和脂质体形态17超微结构的改变。 注:超微结构的改变可以通过图像分析软件进行量化，如ImageJ的13（http://imagej.nih.gov/ij/）。蛋白质装修时，可以用肉眼脂模板相比，来衡量。另外，管状段的直径可以从组件13的最外部分来测量。可使用低温电子显微镜17进行更为详细的分析。这种方法可用于图像天然蛋白质 - 类脂组件在溶剂而不使用重金属污渍的涂层样品。以这种方式，详细的结构特征在负染色，包括在底层脂质形态的改变并不明显，可以检查和定量。 </li></ol></li></ol> 3.使用脚手架脂质体对酶法测定注意:COLORI公制GTP酶测定18用于测量通过GTP水解磷酸解放。替代GTP酶测定法是可用的19，并且可以根据需要来实现。 <ol><li>孵育他的标记MffΔTM（MFF），Fis1ΔTM（FIS1）或GFP（5微米决赛全部）配有支架脂质体（150μM最终）在RT在缓冲液A + BME（体积= 30μL）15分钟。添加DRP1（500nM终），并在RT（体积= 80微升）孵育另外的15分钟。 注:FIS1被以类似的方式向MFF 2纯化，但是省略了离子交换层析步骤。 His标记的GFP的目的是屏蔽NTA（镍2+）头部基团，并防止与其他蛋白质的非特异性电荷相互作用。如果没有效果是在不存在的GFP观察到的，则该控制可能不需要。替代阻断蛋白质（的大小相当于感兴趣的蛋白）可以使用为好，但绿色荧光蛋白允许与SCAF的相互作用的直接可视化折叠脂质体。 </li><li>传输管以热循环设置为37℃，并开始通过加入GTP和的 MgCl 2反应（1分别毫和2mM终;体积= 120微升）。 </li><li>在所需的时间点（ 即 T = 5，10，20，40，60分钟），转移反应的20微升含0.5M的EDTA 5微升以螯合镁离子和停止反应微量滴定板的孔中。 </li><li>在缓冲区A + BME稀释KH 2 PO 4校准结果准备一套磷酸标准。的标准的一个组有用是100，80，60，40，20，10，5，和0微米。添加各20μL的于含0.5M的EDTA 5微升孔中。 </li><li>添加150孔雀石μL的绿色试剂（1毫孔雀绿甲醇，10毫钼酸铵四水合物，和1N HCl中）到每个孔中，并读取OD加入后650 5分钟。 注:GTP是酸不稳定的，并且将在孔雀绿试剂的存在下水解。可以保证T他补充孔雀石试剂和阅读之间的时间是恒定的，以确保可重复的结果。 </li><li>产生通过绘制标准的OD 650作为磷酸盐浓度的函数的标准曲线。使用线性回归来确定样品中的OD 650和磷酸盐浓度之间的关系。 </li><li>使用线性回归，转换蛋白反应样品的OD 650μM磷酸盐。通过标绘磷酸盐浓度作为时间的函数确定磷酸盐一代的各反应混合物中的速率，并转换由DRP1浓度（0.5μM）除以速率为k 猫 。 注意:只有在初始线性速率应被用来确定磷酸产生的速率，并且必须使用至少3个数据点。如果反应的速率足够快，以致前三个数据点不是线性的（ 即线性拟合的R 2是小于0.9）的标志应进行至少3个时间点ificantly较短的时间过程。 </li></ol>

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A., Poolman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+diffusion+of+membrane+proteins.">Lateral diffusion of membrane proteins.</a> J Am Chem Soc. 131 (35), 12650-12656 (2009).</li><li>Gambin, Y., Reffay, M., 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=Variation+of+the+lateral+mobility+of+transmembrane+peptides+with+hydrophobic+mismatch.">Variation of the lateral mobility of transmembrane peptides with hydrophobic mismatch.</a> J Phys Chem. B. 114 (10), 3559-3566 (2010).</li><li>Wilson-Kubalek, E. M., Brown, R. E., Celia, H., Milligan, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+nanotubes+as+substrates+for+helical+crystallization+of+macromolecules.">Lipid nanotubes as substrates for helical crystallization of macromolecules.</a> Proc Natl Acad Sci. 95 (14), 8040-8045 (1998).</li></ol>

The authors have nothing to disclose.

这个协议提供了调查涉及整合膜蛋白的蛋白质 - 蛋白质相互作用的方法。利用模块化脂质体脚手架，调查能够评估脂质近侧环境的一种或多种蛋白质的活性。以前的研究已经证明了质膜24的受体酶类似的方法- 26。我们扩大了这种方法纳入脂质辅因子和探索弥补线粒体分裂机械mechanoenzymatic核心蛋白之间的相互作用。 对于以上提出的模型系统中，我们发?...

研究膜近端蛋白质-蛋白质相互作用是一个具有挑战性的努力由于在扼要涉及1的整合膜蛋白的天然环境中的困难。这是由于洗涤剂增溶的必要性和蛋白在脂蛋白体不一致取向。为了避免这些问题，我们采用的策略，从而整合膜蛋白的可溶性结构域被表达为His-标签的融合蛋白质，以及这些的可溶性片段在脂质经由相互作用锚定到支架的脂质体与NTA（镍2+）首基表面。使用这些支架，脂质 - 近侧蛋白质相互作用可以在一定范围内的脂质和蛋白质的组合物进行研究。 我们已经有效地应用这种方法来调查支配线粒体裂变复杂的装配关键蛋白 - 蛋白相互作用并检查调节这种公关脂相互作用ocess 2。期间线粒体裂变，一个保守的膜重塑的蛋白质，称为动力素相关蛋白1（DRP1）3，响应于调节能量平衡，凋亡信号等几个蜂窝信号募集至线粒体外膜（OMM）的表面整体线粒体的过程。 8 -这个大，胞浆GTP酶是通过与不可分割的OMM蛋白4次互动招募到线粒体的表面。一个这样的蛋白质的作用，线粒体裂变因子（MFF），一直难以阐明由于在体外用DRP1表观弱相互作用。然而，遗传研究清楚地表明，MFF是成功的线粒体分裂7,8至关重要。在这个手稿中描述的方法能够通过引入促进DRP1-MFF相互作用同时脂质相互作用克服了以前的缺点。总体而言，这一新的分析revea导致基本相互作用指导线粒体分裂复杂的装配和这一重要分子机器正在进行的结构和功能研究提供了一个新的阶段。 迄今为止，DRP1以及MFF之间的相互作用的研究已经通过MFF 9固有的灵活性复杂，DRP1的异质性聚合物2,10，并且难以纯化和重建全长MFF具有完整跨膜结构域11。我们通过使用NTA（镍离子 ）支架脂质体重组His标记的MFF缺乏跨膜结构域（MffΔTM-他6）解决了这些难题。这一战略是有利的，因为MffΔTM是非常易溶当E的表达。大肠杆菌 ，这种分离蛋白很容易在支架脂质体重组。当拴这些脂质模板，MFF假定在膜的表面上的相同的，面向外的取向。除了这些优点，线粒体脂质，如心磷脂，分别加入稳定MFF折叠和关联与膜11。心磷脂也与DRP1 2,12的可变结构域可稳定这种无序区和促进裂变机械的组件交互。 这种稳健的方法可广泛应用于未来的研究，设法评估近膜蛋白相互作用。通过使用附加的系链/亲和力的相互作用，这些膜重建研究的复杂性可以提高，以模仿附加在细胞内的膜的表面发现的复杂性。与此同时，脂质组合物可以被修改以更准确地模仿这些大分子复合的天然环境。总之，这种方法提供了研究蛋白质的关键的细胞PROC中的相对贡献和脂类塑造膜形态为手段S弯。

<table><tbody><tr><td>Phosphatidylcholine (DOPC)</td><td>Avanti Polar Lipids</td><td>850375</td><td></td></tr><tr><td>Phosphatidylethanolamine (DOPE)</td><td>Avanti Polar Lipids</td><td>850725</td><td></td></tr><tr><td>DGS-NTA(Ni&lt;sup&gt;2+&lt;/sup&gt;)</td><td>Avanti Polar Lipids</td><td>790404</td><td></td></tr><tr><td>Bovine Heart Cardiolipin (CL)</td><td>Avanti Polar Lipids</td><td>840012</td><td></td></tr><tr><td>Chloroform</td><td>Acros Organics</td><td>268320010</td><td></td></tr><tr><td>Liposome Extruder</td><td>Avanti Polar Lipids</td><td>610023</td><td></td></tr><tr><td>Cu/Rh Negative Stain Grids</td><td>Ted Pella</td><td>79712</td><td></td></tr><tr><td>Microfuge Tube</td><td>Beckman</td><td>357448</td><td></td></tr><tr><td>GTP</td><td>Jena Biosciences</td><td>NU-1012</td><td></td></tr><tr><td>GMP-PCP</td><td>Sigma Aldrich</td><td>M3509</td><td></td></tr><tr><td>Microtiter Plate strips</td><td>Thermo Scientific</td><td>469949</td><td></td></tr><tr><td>EDTA</td><td>Acros Organics</td><td>40993-0010</td><td></td></tr><tr><td>Instant Blue Coomassie Dye</td><td>Expedeon</td><td>ISB1L</td><td></td></tr><tr><td>HEPES</td><td>Fisher Scientific</td><td>BP310</td><td></td></tr><tr><td>BME</td><td>Sigma Aldrich</td><td>M6250</td><td></td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P330</td><td></td></tr><tr><td>KOH</td><td>Fisher Scientific</td><td>P250</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Acros Organics</td><td>223211000</td><td></td></tr><tr><td>4 - 20% SDS-PAGE Gel</td><td>Bio Rad</td><td>456-1096</td><td></td></tr><tr><td>4x Laemmli Loading Dye</td><td>Bio Rad</td><td>161-0747</td><td></td></tr><tr><td>HCL</td><td>Fisher Scientific</td><td>A144S</td><td></td></tr><tr><td>Malachite Green Carbinol</td><td>Sigma Aldrich</td><td>229105</td><td></td></tr><tr><td>Ammonium Molybdate Tetrahydrate</td><td>Sigma Aldrich</td><td>A7302</td><td></td></tr><tr><td>Laboratory Film</td><td>Parafilm</td><td>PM-996</td><td></td></tr><tr><td>Uranyl Acetate</td><td>Polysciences</td><td>21447</td><td></td></tr><tr><td>Tecnai T12 100 keV Microscope</td><td>FEI</td><td></td><td></td></tr><tr><td>Optima MAX</td><td>Beckman</td><td></td><td></td></tr><tr><td>TLA-55 Rotor</td><td>Beckman</td><td></td><td></td></tr><tr><td>Refrigerated CentriVap Concentrator</td><td>Labconico</td><td></td><td></td></tr><tr><td>Mastercycler Pro Thermocycler</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>VersaMax Microplate reader</td><td>Molecular Devices</td><td></td><td></td></tr></tbody></table>

using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro

体外整合膜蛋白的研究经常由疏水跨膜结构域的存在复杂。这些研究进一步复杂化，洗涤剂溶解的膜蛋白的重新纳入到脂质体是一种随机过程，其中蛋白质拓扑是无法执行。本文提供了一种替代方法，它利用基于脂质体的脚手架这些具有挑战性的技术。蛋白质溶解度是由跨膜结构域的缺失增强，并且这些氨基酸与一个系栓部分取代，如His标签。此系链与，这在脂质体的表面实施均匀的蛋白质拓扑结构（由次氮基三乙酸（NTA（镍2+））为His-标记的蛋白的协调的Ni 2+）的锚定基团相互作用。一个例子给出，其中用完整的膜蛋白，线粒体裂变因子（MFF）动力素相关蛋白1（DRP1）之间的相互作用，是英伟使用这种支架脂质体法stigated。在这项工作中，我们已经证明MFF的有效招募可溶性DRP1到脂质体的表面上，这刺激其GTP酶活性的能力。此外，DRP1能够tubulate在特定脂质的存在下MFF装饰脂质模板。本实施例说明采用结构和功能分析的脂质体的支架的有效性，并突出MFF在调节DRP1活性的作用。

而DRP1和MFF之间的相互作用已被证明是线粒体裂变重要，这种相互作用已经难以在体外概括。我们的目标是更好地模拟蜂窝环境，其中DRP1以及MFF相互作用。为此目的，含有NTA（镍2+）头部基团的非限制性浓度的脂质体通过如上所述再水化脂质膜制备。脂质溶液最初由异质直径单层和多层囊泡的由溶液（ 图1a）的不透明度为证。此不透明度通过冻?...

Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

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

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

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 ...

using-scaffold-liposomes-to-reconstitute-lipid-proximal-protein

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

8.8K 次观看.  Case Western Reserve University School of Medicine. This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment. 

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

Detergent-free Ultrafast Reconstitution of Membrane Proteins into Lipid Bilayers Using Fusogenic Complementary-charged Proteoliposomes.

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents.
Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged. We also show how to reconstitute membrane proteins into fusogenic proteoliposomes with a fast 30-min protocol.
Combining these two approaches, we demonstrate a fast assembly of an electron transport chain consisting of two membrane proteins from E. coli, a primary proton pump bo3-oxidase and F1Fo ATP synthase, in membranes of vesicles of various sizes, ranging from 0.1 to &#62;10 microns, as well as ATP production by this chain.

Here we present two ultrafast protocols for reconstitution of membrane proteins into fusogenic proteoliposomes, and fusion of such proteoliposomes with target lipid bilayers for detergent-free delivery of these membrane proteins into the postfusion bilayer. The combination of these approaches enables fast and easily controlled assembly of complex multi-component membrane systems.

无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers ...

科研

生物化学

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

A Model Membrane Platform for Reconstituting Mitochondrial Membrane Dynamics

Mitochondrial dynamics is essential for the organelle&#8217;s diverse functions and cellular responses. The crowded, spatially complex, mitochondrial membrane is a challenging environment to distinguish regulatory factors. Experimental control of protein and lipid components can help answer specific questions of regulation. Yet, quantitative manipulation of these factors is challenging in cellular assays. To investigate the molecular mechanism of mitochondria inner-membrane fusion, we introduced an in vitro reconstitution platform that mimics the lipid environment of the mitochondrial inner-membrane. Here we describe detailed steps for preparing lipid bilayers and reconstituting mitochondrial membrane proteins. The platform allowed analysis of intermediates in mitochondrial inner-membrane fusion, and the kinetics for individual transitions, in a quantitative manner. This protocol describes the fabrication of bilayers with asymmetric lipid composition and describes general considerations for reconstituting transmembrane proteins into a cushioned bilayer. The method may be applied to study other membrane systems.

Mitochondrial fusion is an important homeostatic reaction underlying mitochondrial dynamics. Described here is an in vitro reconstitution system to study mitochondrial inner-membrane fusion that can resolve membrane tethering, docking, hemifusion, and pore opening. The versatility of this approach in exploring cell membrane systems is discussed.

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 ...

Cell-Free Production of Proteoliposomes for Functional Analysis and Antibody Development Targeting Membrane Proteins

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug discovery because they are the targets of more than half of all drugs. An obstacle to conducting biochemical, biophysical, and structural studies of membrane proteins as well as antibody development has been the difficulty in producing large amounts of high-quality membrane protein with correct conformation and activity. Here we describe a &#8220;bilayer-dialysis method&#8221; using a wheat germ cell-free system, liposomes, and dialysis cups to efficiently synthesize membrane proteins and prepare purified proteoliposomes in a short time with a high success rate. Membrane proteins can be produced as much as in several milligrams, such as GPCRs, ion channels, transporters, and tetraspanins. This cell-free method contributes to reducing the time, cost and effort for preparing high-quality proteoliposomes, and provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

This protocol describes an efficient cell-free method for production of high-quality proteoliposome by bilayer-dialysis method using wheat cell-free system and liposomes. This method provides suitable means for functional analysis of membrane proteins, drug targets screening, and antibody development.

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

Membrane proteins play essential roles in a variety of cellular processes and perform vital functions. Membrane proteins are medically important in drug ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

在膜上重构隔膜组件以研究生物物理性质和功能

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

用于复杂蛋白质网络的脂质囊泡包封方案

Construction of Out&#45;of&#45;Equilibrium Metabolic Networks in Nano&#45; and Micrometer&#45;Sized Vesicles

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are&#160;trapped in microfluidic devices for analysis by optical microscopy.

作者聚焦:应对合成细胞工程中的挑战

We present a protocol for reconstituting membrane proteins and encapsulating enzymes and other water-soluble components in lipid vesicles of sub-micrometer and micrometer size.

在纳米和微米大小的囊泡中构建失衡代谢网络

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, ...

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

神经科学

Assembly of Cell Mimicking Supported and Suspended Lipid Bilayer Models for the Study of Molecular Interactions

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial protective barrier for all cell types, separating the internal cellular components from the extracellular environment. These membranes are composed largely of a lipid bilayer, which contains outer hydrophilic head groups and inner hydrophobic tail groups, along with various proteins and cholesterol. The composition and structure of the lipids themselves play a crucial role in regulating biological function, including interactions between cells and the cellular microenvironment, which may contain pharmaceuticals, biological toxins, and environmental toxicants. In this study, methods to formulate uni-lipid and multi-lipid supported and suspended cell mimicking lipid bilayers are described. Previously, uni-lipid phosphatidylcholine (PC) lipid bilayers as well as multi-lipid placental trophoblast-inspired lipid bilayers were developed for use in understanding molecular interactions. Here, methods for achieving both types of bilayer models will be presented. For cell mimicking multi-lipid bilayers, the desired lipid composition is first determined via lipid extraction from primary cells or cell lines followed by liquid chromatography-mass spectrometry (LC-MS). Using this composition, lipid vesicles are fabricated using a thin-film hydration and extrusion method and their hydrodynamic diameter and zeta potential are characterized. Supported and suspended lipid bilayers can then be formed using quartz crystal microbalance with dissipation monitoring (QCM-D) and on a porous membrane for use in a parallel artificial membrane permeability assay (PAMPA), respectively. The representative results highlight the reproducibility and versatility of in vitro cell membrane lipid bilayer models. The methods presented can aid in rapid, facile assessment of the interaction mechanisms, such as permeation, adsorption, and embedment, of various molecules and macromolecules with a cell membrane, helping in the screening of drug candidates and prediction of potential cellular toxicity.

This protocol describes the formation of cell mimicking uni-lipid and multi-lipid vesicles, supported lipid bilayers, and suspended lipid bilayers. These in vitro models can be adapted to incorporate a variety of lipid types and can be used to investigate various molecule and macromolecule interactions.

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究

Model cell membranes are a useful screening tool with applications ranging from early drug discovery to toxicity studies. The cell membrane is a crucial ...

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用

摘要

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无洗涤剂超快重组膜蛋白成脂双层使用融合补充充电 Proteoliposomes。

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

重建线粒体膜动力学的模型膜平台

无细胞生产蛋白脂质体，用于功能分析和靶向膜蛋白的抗体开发

在膜上重构隔膜组件以研究生物物理性质和功能

在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

在纳米和微米大小的囊泡中构建失衡代谢网络

单脂蛋白的SNARE介导融合在一个微流控流细胞拴支持双分子层通过偏光显微镜TIRF监控

细胞模拟支持和悬浮脂质双分子层模型的组装用于分子相互作用研究