Name: using scaffold liposomes to reconstitute lipid-proximal protein-protein interactions in vitro
Uploaded: 2017-01-11T12:30:00
Description: Watch this Scientific Journal Video about Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro at JoVE.com

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?...

<ol>
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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 border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylcholine (DOPC)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850375</td>
			<td style="width:72px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Phosphatidylethanolamine (DOPE)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">850725</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:287px;">DGS-NTA(Ni2+)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">790404</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Bovine Heart Cardiolipin (CL)</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">840012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Chloroform</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">268320010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Liposome Extruder</td>
			<td style="width:229px;">Avanti Polar Lipids</td>
			<td style="width:136px;">610023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Cu/Rh Negative Stain Grids</td>
			<td style="width:229px;">Ted Pella</td>
			<td style="width:136px;">79712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microfuge Tube</td>
			<td style="width:229px;">Beckman</td>
			<td style="width:136px;">357448</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GTP</td>
			<td style="width:229px;">Jena Biosciences</td>
			<td style="width:136px;">NU-1012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">GMP-PCP</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M3509</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Microtiter Plate strips</td>
			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:136px;">469949</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">EDTA</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">40993-0010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">Instant Blue Coomassie Dye</td>
			<td style="width:229px;">Expedeon</td>
			<td style="width:136px;">ISB1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">HEPES</td>
			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">BP310</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">BME</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
			<td></td>
		</tr>
		<tr height="21">
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</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 ...

The overall goal of this technique is to capture lipid-proximal protein-protein interactions in vitro. This method can help answer questions in the mitochondrial dynamics field, such as how protein-protein interactions at the mitochondrial outer membrane can be affected by accessory lipids. The main advantage of this technique is that tethering soluble protein domains to lipid templates ensures that a more native confirmation can be achieved.Moreover, complex interactions between soluble proteins and their tethered partners can be assessed in the presence of defined lipid cofactors. Though we use this method to investigate protein interactions in the mitochondrial fission machinery, it can also be applied to other protein complexes that localize to diverse membrane compartments. Demonstrating this procedure will be Ryan, a grad student from my laboratory.First, combine chloroform solutions of the desired lipids in a clean glass test tube. Evaporate the solvent with dry nitrogen gas while rotating the tube to form a thin lipid film. Then remove the residual solvent with the centrifugal evaporator for one hour at 37 degrees Celsius.Following this, add pre-heated buffer A to give a final lipid concentration of one to two millimolar. Incubate for 30 minutes at 37 degrees Celsius with occasional vortexing to fully resuspend the lipid mixture. After incubation, transfer the lipid mixture to a plastic test tube and place the tube in liquid nitrogen for approximately 30 seconds until completely frozen.Then place the tube in a 37 degree Celsius waterbath for one to two minutes until fully thawed. Next prepare a lipid extruder by soaking four filter supports and a polycarbonate filter in buffer A and assembling the extruder according to the manufacturer's instructions. Extrude the lipid solution through the filter 21 times using gentle constant pressure to ensure a homogenous size distribution.While extruding, use slow constant pressure to ensure liposome size homogeneity. With a one micron filter, this is less important. But with smaller filter sizes, can introduce more apparent heterogeneity.Store the extruded liposomes at four degrees Celsius. Incubate His-tagged mitochondrial fission factor or Mff with scaffold liposomes for at least 15 minutes at room temperature in buffer A and BME. For an Mff-free control, incubate the liposomes with a His-tagged control protein, such as GFP, to bind and shield exposed Nickel-NTA.Add dynamin-related protein one or Drp1 to the liposome mixture and incubate for one hour at room temperature. Next, transfer five microliters of the sample to a sheet of laboratory film and lay a carbon-coated copper-rhodium grid on top of the sample. After incubating for one minute, remove the excess liquid with filter paper and add a drop of 2%uranyl acetate.After incubating for another minute, remove the excess stain with filter paper, and transfer the sample to a grid box. On the following day, image the samples using a transmission electron microscope at the 18, 500 to 30, 000X magnification to observe ultrastructural changes in protein and liposome morphologies. Incubate His-tagged Mff and Fis1 with the scaffold liposomes for 15 minutes at room temperature in buffer A and BME.Add Drp1 and incubate each mixture for an additional 15 minutes at room temperature. Transfer the tubes to the thermocycler set to 37 degrees Celsius and initiate the reactions by adding GTP and magnesium chloride. At the desired time points, transfer 20 microliters of each reaction mixture to wells of a microtiter plate containing five microliters of 0.5 molar EDTA to chelate the magnesium and stop the reaction.Following this, prepare a set of phosphate standards by diluting potassium phosphate in buffer A and BME to calibrate the results. Add 20 microliters of each standard to the wells containing five microliters of 0.5 molar EDTA. Add 150 microliters of malachite green reagent to each well.Finally, read the OD 650 five minutes after each addition. GTP will hydrolyze slowly in the presence of the acidic malachite green reagent. So this reagent should be added to all sample wells within 10 minutes of measuring the absorbance to maximize signal to noise.In the absence in Drp1, neither Mff nor GFP resulted in membrane deformation. And only featureless liposomes were observed for GFP-decorated enriched scaffold liposomes or ESL. However, when Drp1 was added to Mff-decorated ESL templates remodeling of the liposomes was evident.Decoration of the SL with Mff enhanced GTPase activity. Conversely, when the exposed NTA head groups were blocked with His-tagged GFP, this augmented GTPase activity was ablated. Tethering of Fis1 lacking its transmembrane domain to the SL, also failed to elicit a stimulation of Drp1's GTPase activity.Similar to the SL, addition of SL/Cl to Drp1 resulted in a slight stimulation of GTPase activity that was reversed by tethering His-tagged Fis1 or GFP to the liposomes. A synergy between Mff and cardiolipin was observed as the GTPase activity of Drp1 was stimulated 2.6-fold when it was incubated with Mff-decorated SL/CL. When SL/PE/CL templates were decorated with Mff, Drp1 activity was enhanced.The ability of Drp1 to remodel liposomes into lipid tubules was enhanced by the addition of PE to the scaffold liposomes. Once mastered, these scaffolds can be prepared and used in hours if the procedure is performed properly. Following this procedure, other protein and lipid interaction methods like sedimentation and light-scattering assays can be performed in order to characterize functional interactions.The application of this technique allows people examining protein translocation to a defined biological membrane to explore transient protein interactions and the impact of specific lipid cofactors in the formation and stabilization of essential cellular assemblies.

Research

JoVE Journal

Biochemistry

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

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

时间分辨电喷雾电离氢氘交换质谱用于研究蛋白质结构与动态

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Biotinylated Cell-penetrating Peptides to Study Intracellular Protein-protein Interactions

化细胞穿透肽研究胞内蛋白-蛋白质相互作用

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

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 ...

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

Site-Directed Mutagenesis for In Vitro and In Vivo Experiments Exemplified with RNA Interactions in Escherichia Coli

体外和体内实验的位定向诱变-以大肠杆菌rna相互作用为例

Site-directed mutagenesis is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

生物层干涉测量(BLI)在转录中研究蛋白质-蛋白质相互作用的应用

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Evaluation of Protein&#8211;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 ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...