Name: genetically-encoded molecular probes to study g protein-coupled receptors
Uploaded: 2013-09-13T16:30:00
Description: Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

我们感谢慷慨支持的几个基金会和慈善捐赠（见SakmarLab.org）。

1。非天然氨基酸位点特异性基因掺入到G蛋白偶联受体<ol><li>保持HEK293T细胞在DMEM（4.5 g / L的葡萄糖，2mM谷氨酰胺）中在37℃下在5％CO 2气氛中添加了10％胎牛血清（FBS）。 </li><li>转染生长至在使用Lipofectamine试剂加有10厘米板60-80％汇合的细胞。 <ol><li>到750微升DMEM中，加入10微升加试剂，3.5微克的GPCR的cDNA（pMT4.的Rho或染pcDNA3.1。CCR5）含有琥珀终止密码子在所希望的位置上，3.5微克的抑制基?...

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在这里，我们描述了一个完善的方法进行位点特异性结合反应的探针，AZF，进入G蛋白偶联受体，并证明这个工具来研究G蛋白偶联受体的结构和动力学的三个有用的应用程序。我们的方法来位点特异性地并入UAAs规避与基于化学标记32,33或附加光交联剂34到单个可访问的半胱氨酸突变体的替代策略的一个基本问题。虽然，针对半胱氨酸硫醇基团的化学已被用于附加的荧光探针，这种?...

七螺旋G蛋白偶联受体（GPCR）包括高度动态的膜蛋白介导的重要和多样的细胞外信号的超家族。在古典范式，受体激活耦接配体诱导的构象变化。1，在G蛋白偶联受体的结构生物学2最近的进步已经提供的跨膜信号转导的分子机制显著洞察力。3-5然而，为了理解具有更大的化学精度的作用机理和G蛋白偶联受体信号传导的结构动力学，办法的工具包，需要把信息分子和化学探针审问积极信号复合体。 为此，我们适应的方法来位点特异性地引入基于非天然氨基酸（UAA）的诱变使用琥珀密码子之前由舒尔茨和c开创抑制技术非或最低限度地扰动探测到表达的受体oworkers 6我们优化了UAA诱变的方法来实现对蛋白质，如G蛋白偶联受体，它们是难以表达，在大多数的异源系统比哺乳动物细胞等的高产量表达和诱变系统。使用正交设计的抑制性tRNA和进化氨酰-tRNA合成酶对用于特定UAA，我们位点特异性地引入UAAs中表达靶GPCR的。 UAAs的成功的掺入， 对 -乙酰基-L-苯丙氨酸（ACF）， 对 -苯甲酰基-L-苯丙氨酸（BZF），和p-叠氮基-L-苯丙氨酸（AZF）已被证明在我们的模型中的GPCR -视紫红质和人类CC趋化因子受体，CCR5 7,8 原则上，UAA可以基因编码的蛋白质序列中的任何位置，这个属性是一个非常宝贵的生化工具，因为它使一个目标GPCR的单密码子扫描。我们特别关注这里的UAA，AZF，其中有一个反应阿紫的多功能性做的部分。除了 ​​作为一个独特的红外（IR）探测器，8,9 AZF也可以作为一个可光活化交联剂通过与邻近的伯胺或脂族氢的反应。此外，生物惰性叠氮基可以参与作为生物正交标记反应选择性的化学处理。在这里，我们提出了举例说明位点特异性结合AZF的有用的应用程序转换为G蛋白偶联受体，例如有针对性的光交诱捕受体 - 配体复合物，并通过G蛋白偶联受体抗原生物正交标记和荧光标记的策略进行修改。 光活化试剂已被用于研究生物系统自1960年以来，10在这期间，受体-配体的交联实验丰盈已报道研究G蛋白偶联受体复合物，其中大部分涉及利用光亲和配体。11,12然而，这些应用技术上的限制，因为它们必需参数E的合成配体带有交联基团的13-15再者，在配位体的交联基团，它是具有挑战性的，以确定在G蛋白偶联受体的交联点的位置。位点特异性地引入光交联基团如UAAs到使用琥珀密码子抑制技术的蛋白质是一种有价值的进步。16,17我们开发了一种光致交联的技术，以确定对涉及的受体-配体复合物的形成是一个受体的结合界面通过引入光不稳定基团引入GPCR的活细胞。18,19在这里，我们描述的实验方案和数据分析的方法，应用该目标光交联技术，以确定一个小分子配体的结合位点，氚标记马拉韦罗，对CCR5。该方法利用了对配体的放射性的手柄，除了保留配体的天然化学结构的精确定量。 基于荧光技术直接探测受体的构象状态支持受体激活的结构基础的精确理解。20,21然而，拥有技术引进荧光标记成G蛋白偶联受体的灵活性，位点特异性是有限的。我们感兴趣的是使用生物正交化学修饰策略，以促进G蛋白偶联受体信号复合物的单分子检测（SMD），22叠氮基可以参与生物正交化学物质，如施陶丁格-Bertozzi教授结扎，23，24无铜应变推动叠氮炔环加成反应（SpAAC）25和铜催化的叠氮-炔环加成反应（CuAAC）26在这里，我们重点放在涉及叠氮化物和磷化氢之间的特异性反应的施陶丁格-Bertozzi教授连接反应。我们展示了使用两种不同的膦衍生物，缀合的肽表位（FLAG肽）或荧光标记的（荧光素），以实现GPCR的位点特异性修饰。 我们以前优化的视紫红质AZF变种利用X-射线晶体结构和动态模拟来选择是溶剂暴露目标站点的站点特定标记的条件。27，28，我们也说明了实现无背景标签的可行性，施陶丁格- Bertozzi教授结扎29我们在这里展示用于实现该被固定在免疫亲和基质，并随后通过在凝胶荧光可视化的洗涤剂增溶的受体荧光标记的一般方法。此外，我们证明这个标签战略，以确定适合于对未知结构，CCR5的受体标记位置的有用的扩展。这是通过使用一个依赖于的AZF-GPCR在基于细胞的半高通量形式的变体生物正交变型的靶向肽的表位标记的战略。30这方法利用细胞表面的ELISA的多步骤的检测性能监视标记的事件。 我们在这里讨论的方法是通用的，原则上可以适用于使用琥珀密码子抑制技术与AZF注册成立的任何G蛋白偶联受体。在这里给出的细节，我们涉及使用非天然氨基酸的诱变方法和其后续的应用，以促进G蛋白偶联受体的结构和动力学研究UAAs如AZF结合受体的哺乳动物细胞表达的步骤的协议。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmid pSVB. Yam</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2008)</td>
		</tr>
		<tr>
			<td>Plasmid pcDNA.RS for azF</td>
			<td></td>
			<td></td>
			<td>(Ye et al., 2009)</td>
		</tr>
		<tr>
			<td>Plasmids pMT4.Rho and pcDNA 3.1.CCR5</td>
			<td></td>
			<td></td>
			<td>Optionally contain the amber stop codon (TAG) at a desired position</td>
		</tr>
		<tr>
			<td>HEK 293T cells</td>
			<td></td>
			<td></td>
			<td>Adherent cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Gibco</td>
			<td>10566</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14200</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gemini Bio-products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine Plus</td>
			<td>Invitrogen</td>
			<td>Lipofectamine: 18324-012 
			Plus: 11514-015</td>
			<td></td>
		</tr>
		<tr>
			<td>p-azido-L-phenylalanine</td>
			<td>Chem-Impex International</td>
			<td>6162</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 1. Site-specific genetic incorporation of unnatural amino acids into GPCRs materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Maxima ML-3500S UV-A lamp</td>
			<td>Spectronics Corporation</td>
			<td></td>
			<td>azF is activated by 365-nm light</td>
		</tr>
		<tr>
			<td>Hank&#39;s Buffered Salt Solution (HBSS)</td>
			<td>Gibco</td>
			<td>14065</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Irvine Scientific</td>
			<td>9319</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Roche</td>
			<td>3117405001</td>
			<td></td>
		</tr>
		<tr>
			<td>Tritium-labeled ligand</td>
			<td></td>
			<td></td>
			<td>From collaborator (Grunbeck et al., 2012)</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>1% (w/v) sodium dodecyl sulfate (SDS)</td>
			<td>Fisher Scientific</td>
			<td>BP166</td>
			<td></td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Trans-Blot SD apparatus</td>
			<td>Biorad</td>
			<td></td>
			<td>Apparatus for semi-dry transfer</td>
		</tr>
		<tr>
			<td>Immobilon polyvinylidene difluoride (PVDF) membrane</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Non-fat powered milk</td>
			<td>Fisher Scientific</td>
			<td>NC9934262</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Aldrich</td>
			<td>274348</td>
			<td></td>
		</tr>
		<tr>
			<td>Ecoscint A</td>
			<td>National Diagnostics</td>
			<td>LS-273</td>
			<td>Scintillation fluid</td>
		</tr>
		<tr>
			<td>Scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>333726</td>
			<td></td>
		</tr>
		<tr>
			<td>LKB Wallac 1209 Rackbeta Liquid Scintillation Counter</td>
			<td>Perkin Elmer</td>
			<td></td>
			<td>Beta-Scintillation counter</td>
		</tr>
		<tr>
			<td>Anti-rhodopsin 1D4 mAb</td>
			<td>National Cell Culture Center</td>
			<td>custom</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated anti-mouse IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperSignal West Pico Chemiluminescent Substrate</td>
			<td>Thermo Scientific</td>
			<td>34080</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyblot CL AR film</td>
			<td>Denville</td>
			<td>E3018</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 2. Targeted photocrosslinking materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Invitrogen</td>
			<td>15050065</td>
			<td></td>
		</tr>
		<tr>
			<td>FLAG-triarylphosphine</td>
			<td>Sigma</td>
			<td>GPHOS1</td>
			<td></td>
		</tr>
		<tr>
			<td>M2 FLAG mAb</td>
			<td>Sigma</td>
			<td>F1804</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-CCR5 2D7 mAb</td>
			<td>BD Biosciences</td>
			<td>555990</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-lysine</td>
			<td>Sigma</td>
			<td>P6407</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Costar</td>
			<td>3601</td>
			<td>Clear bottom, high binding EIA/RIA</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (Calcium, Magnesium)</td>
			<td>Gibco</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>16% Paraformaldehyde</td>
			<td>EMS</td>
			<td>28908</td>
			<td></td>
		</tr>
		<tr>
			<td>HRP-conjugated</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit IgG</td>
			<td>KPL, Inc.</td>
			<td>474-1516</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplex Red</td>
			<td>Invitrogen</td>
			<td>A12222</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen peroxide</td>
			<td>DE Healthcare Products</td>
			<td>97-93399</td>
			<td></td>
		</tr>
		<tr>
			<td>CytoFluor II fluorescence multi-well plate reader</td>
			<td>Perseptive Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 3. Targeted peptide-epitope tagging and cell surface ELISA materials</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>[header]</td>
		</tr>
		<tr>
			<td>Fluorescein-phosphine</td>
			<td></td>
			<td></td>
			<td>(Huber et al., submitted 2012)</td>
		</tr>
		<tr>
			<td>Nonapeptide (C9 peptide)</td>
			<td>AnaSpec</td>
			<td>62190</td>
			<td>Peptide mimicking the 1D4-epitope NH2-TETSQVAPA-COOH</td>
		</tr>
		<tr>
			<td>1% (w/v) n-dodecyl-&#946;-D-maltoside</td>
			<td>Anatrace</td>
			<td>D310LA</td>
			<td></td>
		</tr>
		<tr>
			<td>1D4-sepharose resin</td>
			<td></td>
			<td></td>
			<td>1D4 mAb immobilized on CNBr-activated sepharose 2B resin</td>
		</tr>
		<tr>
			<td>NuPAGE Novex 4 - 12% SDS gels</td>
			<td>Invitrogen</td>
			<td>NP0322BOX</td>
			<td>SDS-PAGE performed on NuPAGE apparatus</td>
		</tr>
		<tr>
			<td>Confocal Typhoon 9400 fluorescence scanner</td>
			<td>GE Healthcare</td>
			<td>discontinued</td>
			<td>Scanner with 488-nm wavelength laser</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td>Table 4. Fluorescent labeling materials</td>
		</tr>
	</tbody>
</table>

genetically-encoded molecular probes to study g protein-coupled receptors

以促进G蛋白偶联受体（GPCR）信号复合物的结构和动力学研究，新的方法需要引入翔实的探针或标记入表达的受体不扰乱受体的功能。我们使用琥珀密码子抑制技术，基因编码的非天然氨基酸， 对 -叠氮基-L-苯丙氨酸（AZF）在异源表达在哺乳动物细胞中的GPCR各种目标的位置。叠氮基的通用性是这里示出在不同的应用程序来研究G蛋白偶联受体在其天然细胞环境或洗涤剂溶解的条件下进行。首先，我们展示了基于细胞的靶向光交联技术，以确定在G蛋白偶联受体，其中一个氚标记的小分子配体交联到一种基因编码的叠氮基的氨基酸的配体结合口袋的残基。然后，我们通过生物正交的Staudinger-Bertozzi教授结扎反应表明G蛋白偶联受体的位点特异性修饰化用膦衍生物为目标的叠氮基。我们将讨论用于使用全细胞系的ELISA方法在培养及其检测表达的膜蛋白的靶向肽的表位标记的一般策略。最后，我们证明了AZF-G蛋白偶联受体可以选择性标记的荧光探针。讨论的方法是通用的，因为它们可以在原则上被应用到任何的任何氨基酸位置表示的GPCR来询问活性信号传导复合物。

我们所用的非天然氨基酸的诱变方法，以位点特异性地引入分子探针进入的GPCR使用琥珀密码子抑制技术。 图1中的方案概述的显着的步骤的方法，并结合了多功能UAA， 对-叠氮基-L-苯丙氨酸（AZF），进GPCRs的各种应用。的AZF-G蛋白偶联受体在哺乳动物细胞中的表达可让有针对性的光交联配体，并通过生物 ​​正交标记化学针对性的肽表位标记或G蛋白偶联受体的荧光修改。...

Watch this Scientific Journal Video about Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors at JoVE.com

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

在GPCR的各种目标的位置，我们基因编码的非天然氨基酸，对 - 叠氮基-L-苯丙氨酸，并显示在叠氮基团中的不同的应用程序的通用性。这些包括有针对性的光交联技术，以确定在一个G蛋白偶联受体的配体结合口袋的残基，和位点特异性生物正交的GPCR与肽表位标签或荧光探针的修饰。

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

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Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

测试的速度和运动方向的视觉灵敏度在蜥蜴

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

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Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

分离纯化果蝇</em磁性微球的排序>外周神经元

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In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Quantifying Agonist Activity at G Protein-coupled Receptors

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Characterization of G Protein-coupled Receptors by a Fluorescence-based Calcium Mobilization Assay

G蛋白偶联受体的基于荧光的钙动员含量表征

For more than 20 years, reverse pharmacology has been the preeminent strategy to discover the activating ligands of orphan G protein-coupled receptors ...

Visualizing Clathrin-mediated Endocytosis of G Protein-coupled Receptors at Single-event Resolution via TIRF Microscopy

通过全内反射荧光显微镜可视化G蛋白偶联受体的单事件分辨率网格蛋白介导的内吞作用

Many important signaling receptors are internalized through the well-studied process of clathrin-mediated endocytosis (CME). Traditional cell biological ...

Hydroponics: A Versatile System to Study Nutrient Allocation and Plant Responses to Nutrient Availability and Exposure to Toxic Elements

水培：一个多功能的系统来研究营养分配和植物响应养分有效性及暴露于有毒元素

Hydroponic systems have been utilized as one of the standard methods for plant biology research and are also used in commercial production for several ...

Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells

成像G蛋白偶联受体介导的趋化和信令活动在中性粒细胞样细胞HL60

Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many ...