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 border="0" cellpadding="0" cellspacing="0" width="723">
	<tbody>
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			<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>
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		<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>
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			<td height="21" style="height:21px;width:287px;">Chloroform</td>
			<td style="width:229px;">Acros Organics</td>
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			<td style="width:136px;">610023</td>
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			<td height="21" style="height:21px;width:287px;">Cu/Rh Negative Stain Grids</td>
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			<td style="width:136px;">79712</td>
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			<td style="width:229px;">Beckman</td>
			<td style="width:136px;">357448</td>
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			<td style="width:229px;">Thermo Scientific</td>
			<td style="width:136px;">469949</td>
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			<td style="width:136px;">40993-0010</td>
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			<td height="21" style="height:21px;width:287px;">Instant Blue Coomassie Dye</td>
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			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">M6250</td>
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			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P330</td>
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			<td style="width:229px;">Fisher Scientific</td>
			<td style="width:136px;">P250</td>
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			<td height="21" style="height:21px;width:287px;">Magnesium Chloride</td>
			<td style="width:229px;">Acros Organics</td>
			<td style="width:136px;">223211000</td>
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			<td style="width:136px;">161-0747</td>
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			<td height="21" style="height:21px;width:287px;">Malachite Green Carbinol</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">229105</td>
			<td></td>
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			<td height="21" style="height:21px;width:287px;">Ammonium Molybdate Tetrahydrate</td>
			<td style="width:229px;">Sigma Aldrich</td>
			<td style="width:136px;">A7302</td>
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			<td style="width:136px;">21447</td>
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			<td height="21" style="height:21px;width:287px;">Optima MAX</td>
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			<td style="width:136px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:287px;">TLA-55 Rotor</td>
			<td style="width:229px;">Beckman</td>
			<td style="width:136px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:287px;">Refrigerated CentriVap Concentrator</td>
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			<td style="width:136px;"></td>
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		</tr>
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			<td height="21" style="height:21px;width:287px;">Mastercycler Pro Thermocycler</td>
			<td style="width:229px;">Eppendorf</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:287px;">VersaMax Microplate reader</td>
			<td style="width:229px;">Molecular Devices</td>
			<td style="width:136px;"></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

Research

JoVE Journal

Biochemistry

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

8.7K 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

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is necessary to characterize it at the structural and mechanistic level. Several biochemical and biophysical methods exist for such purpose. Among them, fluorescence anisotropy is a powerful technique particularly used when the fluorescence intensity of a fluorophore-labeled protein remains constant upon protein-protein interaction. In this technique, a fluorophore-labeled protein is excited with vertically polarized light of an appropriate wavelength that selectively excites a subset of the fluorophores according to their relative orientation with the incoming beam. The resulting emission also has a directionality whose relationship in the vertical and horizontal planes defines anisotropy (r) as follows: r=(IVV-IVH)/(IVV+2IVH), where IVV and IVH are the fluorescence intensities of the vertical and horizontal components, respectively. Fluorescence anisotropy is sensitive to the rotational diffusion of a fluorophore, namely the apparent molecular size of a fluorophore attached to a protein, which is altered upon protein-protein interaction. In the present text, the use of fluorescence anisotropy as a tool to study protein-protein interactions was exemplified to address the binding between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor like-1 GTPase (EFL1). Conventionally, labeling of a protein with a fluorophore is carried out on the thiol groups (cysteine) or in the amino groups (the N-terminal amine or lysine) of the protein. However, SBDS possesses several cysteines and lysines that did not allow site directed labeling of it. As an alternative technique, the dye 4&#39;,5&#39;-bis(1,3,2 dithioarsolan-2-yl) fluorescein was used to specifically label a tetracysteine motif, Cys-Cys-Pro-Gly-Cys-Cys, genetically engineered in the C-terminus of the recombinant SBDS protein. Fitting of the experimental data provided quantitative and mechanistic information on the binding mode between these proteins.

Protein interactions are at the heart of a cell&#39;s function. Calorimetric and spectroscopic techniques are commonly used to characterize them. Here we describe fluorescence anisotropy as a tool to study the interaction between the protein mutated in the Shwachman-Diamond Syndrome (SBDS) and the Elongation factor-like 1 GTPase (EFL1).

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

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

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

Here we present a protocol to study intracellular protein-protein interactions that is based on the widely used biotin-avidin pull-down system. The modification presented includes the combination of this technique with cell-penetrating sequences. We propose to design cell-penetrating baits that can be incubated with living cells instead of cell lysates and therefore the interactions found will reflect those that occur within the intracellular context. Connexin43 (Cx43), a protein that forms gap junction channels and hemichannels is down-regulated in high-grade gliomas. The Cx43 region comprising amino acids 266-283 is responsible for the inhibition of the oncogenic activity of c-Src in glioma cells. Here we use TAT as the cell-penetrating sequence, biotin as the pull-down tag and the region of Cx43 comprised between amino acids 266-283 as the target to find intracellular interactions in the hard-to-transfect human glioma stem cells. One of the limitations of the proposed method is that the molecule used as bait could fail to fold properly and, consequently, the interactions found could not be associated with the effect. However, this method can be especially interesting for the interactions involved in signal transduction pathways because they are usually carried out by intrinsically disordered regions and, therefore, they do not require an ordered folding. In addition, one of the advantages of the proposed method is that the relevance of each residue on the interaction can be easily studied. This is a modular system; therefore, other cell-penetrating sequences, other tags, and other intracellular targets can be employed. Finally, the scope of this protocol is far beyond protein-protein interaction because this system can be applied to other bioactive cargoes such as RNA sequences, nanoparticles, viruses or any molecule that can be transduced with cell-penetrating sequences and fused to pull-down tags to study their intracellular mechanism of action.

This is a protocol to study intracellular protein-protein interactions based on the biotin-avidin pull-down system with the novelty of combining cell-penetrating sequences. The main advantage is that the target sequence is incubated with living cells instead of cell lysates and therefore the interactions will occur within the cellular context.

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

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated 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 ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the importance of 3-dimensional (3D) cellular assemblies in these fields. Artificial scaffolds have often been used to construct 3D cellular assemblies. However, the scaffolds used to construct cellular assemblies are sometimes toxic and may change the properties of the cells. Thus, it would be beneficial to establish a non-toxic method for facilitating cell-cell contact. In this paper, we introduce a novel method for constructing stable cellular assemblies by using optical tweezers with dextran. One of the advantages of this method is that it establishes stable cell-to-cell contact within a few minutes. This new method allows the construction of 3D cellular assemblies in a natural hydrophilic polymer and is expected to be useful for constructing next-generation 3D single-cell assemblies in the fields of regenerative medicine and tissue engineering.

We demonstrate a novel method for constructing a single-cell-based 3-dimensional (3D) assembly without an artificial scaffold.

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

Here, we present a protocol to produce large amounts of recombinant RNA in Escherichia coli by co-expressing a chimeric RNA that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. The main product is a circular molecule that facilitates purification to homogeneity.

利用病毒衍生系统在大肠杆菌中大规模生产圆形脚手架上的重组 rna

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts ...

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

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

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 is a technique used to introduce specific mutations in DNA to investigate the interaction between small non-coding ribonucleic acid (sRNA) molecules and target messenger RNAs (mRNAs). In addition, site-directed mutagenesis is used to map specific protein binding sites to RNA. A 2-step and 3-step PCR based introduction of mutations is described. The approach is relevant to all protein-RNA and RNA-RNA interaction studies. In short, the technique relies on designing primers with the desired mutation(s), and through 2 or 3 steps of PCR synthesizing a PCR product with the mutation. The PCR product is then used for cloning. Here, we describe how to perform site-directed mutagenesis with both the 2- and 3-step approach to introduce mutations to the sRNA, McaS, and the mRNA, csgD, to investigate RNA-RNA and RNA-protein interactions. We apply this technique to investigate RNA interactions; however, the technique is applicable to all mutagenesis studies (e.g., DNA-protein interactions, amino-acid substitution/deletion/addition). It is possible to introduce any kind of mutation except for non-natural bases but the technique is only applicable if a PCR product can be used for downstream application (e.g., cloning and template for further PCR).

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

Site-directed mutagenesis is a technique used to introduce specific mutations in deoxyribonucleic acid (DNA). This protocol describes how to do site-directed mutagenesis with a 2-step and 3-step polymerase chain reaction (PCR) based approach, which is applicable to any DNA fragment of interest.

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

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 is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

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

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

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

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions largely depend on intracellular protein&#8211;protein interactions. Therefore, research regarding these interactions is indispensable to facilitating the understanding of physiologic processes. Co-precipitation of associated proteins, followed by mass spectrometry (MS) analysis, enables the identification of novel protein interactions. In this study, we have provided details of the novel technique of immunoprecipitation-liquid chromatography (LC)-MS/MS analysis combined with on-membrane digestion for the analysis of protein&#8211;protein interactions. This technique is suitable for crude immunoprecipitants and can improve the throughput of proteomic analyses. Tagged recombinant proteins were precipitated using specific antibodies; next, immunoprecipitants blotted onto polyvinylidene difluoride membrane pieces were subjected to reductive alkylation. Following trypsinization, the digested protein residues were analyzed using LC-MS/MS. Using this technique, we were able to identify several candidate associated proteins. Thus, this method is convenient and useful for the characterization of novel protein&#8211;protein interactions.

Here, we present a protocol for an on-membrane digestion technique for the preparation of samples for mass spectrometry. This technique facilitates the convenient analysis of protein&#8211;protein interactions.

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 ...