Name: purification and reconstitution of trpv1 for spectroscopic analysis
Uploaded: 2018-07-03T13:30:00
Description: Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

我们非常感谢 h. Mchaourab 博士提供了获得 EPR 和鹿分光仪和 t 鲍姆博士的机会, 以提供全长半胱氨酸--较少的 TRPV1 质粒。

1. TRPV1 诱变
注:26的最小 TRPV1 结构是用聚合酶链反应 (PCR) 法从全长半胱氨酸少通道 TRPV127 (图 1) 建立的。这种半胱氨酸的最小 TRPV1 构造 (称为 eTRPV1 以后) 由残留物 110-603 和 627-764 组成。eTRPV1 被克隆在 pMO (一个 pcDNA3.1 的载体) 进行功能分析, 并在一个重组捐助者向量28包含8x 组氨酸-麦芽糖结合蛋白 (MBP)-?...

目前, 哺乳动物膜蛋白的表达和纯化技术使得获得足够数量的蛋白质用于光谱学研究14,15,16,42。在这里, 我们已经适应了这些技术来表达, 纯化, 重建, 并执行光谱分析在 TRPV1。
在《议定书》的关键步骤中, 下面是我们为 TRPV1 诊断的, 可能会对其他蛋白质进行调整。修改 DNA 序列生成?...

近年来, 在单粒子低温电子显微镜 (冷冻 EM) 的研究进展中, 哺乳动物离子通道结构得到了非常的速度。特别是, polymodal 离子通道的结构研究, 如瞬态受体电位辣椒 1 (TRPV1), 已进一步了解其活化机制1,2,3,4,5. 然而, 在膜环境中嵌入离子通道的动态信息需要了解它们的 polymodal 门和药物结合机制。
电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) spectroscopies 提供了一些最明确的机械模型的离子通道6,7,8,9,10,11,12,13. 这些方法主要限于检查原核和 archeal 离子通道, 在细菌抗原时产生大量的洗涤剂纯化蛋白。随着在昆虫和哺乳动物细胞中的真核膜蛋白的发展, 用于功能和结构表征14,15,16, 现在可以获得生物化学用于光谱研究的洗涤剂纯化蛋白质的数量。
EPR 和鹿信号产生于一个 paramagneticspin 标签 (SL) (即methanethiosulfonate) 附着在一个单一的半胱氨酸残留的蛋白质。自旋标签报告三种结构信息: 运动、可访问性和距离。这一信息允许确定残留物是埋藏在蛋白质中, 还是暴露于膜或水环境中的蛋白和配体约束状态13,17,18,19。在高分辨率结构 (如果可用) 的背景下, EPR 和鹿数据提供了在其本机环境中派生动态模型的约束集合, 同时监视可逆的浇口转换 (即闭合、打开、敏化, 和麻木)。此外, 可以通过使用这些环境数据集分配二级结构以及蛋白质20中的位置来获得可能难以由 X 射线晶体学或冷冻 EM 确定的灵活区域。在脂质 nanodiscs 中获得的低温 EM 结构提供了关于离子通道3、21、22、23、24 的浇口的宝贵信息,25;然而, 光谱方法可以提供动态信息从构象状态 (例如, 热变化), 可能是难以确定使用低温 EM。
在去除所有半胱氨酸残留物 (特别是哺乳动物通道中的丰富)、蛋白质产量低、纯化过程中蛋白质不稳定、自旋标记后, 必须克服许多困难, 以实现 EPR 和鹿类, 包括缺乏蛋白质功能。, 以及在洗涤剂或脂质体中的蛋白质聚集。在这里, 我们设计了克服这些关键屏障的协议, 并获得了哺乳动物感官受体的鹿和 EPR 光谱信息。这里的目的是描述的方法, 以表达, 纯化, 标记和重组功能极小的半胱氨酸小鼠 TRPV1 (eTRPV1) 结构的光谱分析。这种方法适用于那些保留其功能的膜蛋白, 尽管除去半胱氨酸残留物或含有半胱氨酸形成二硫键。这些协议的收集可以适应其他哺乳动物离子通道的光谱分析。

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			<td>Fishebrand</td>
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			<td>Drummond Scientific</td>
			<td>3-000-204</td>
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	</tbody>
</table>

purification and reconstitution of trpv1 for spectroscopic analysis

Polymodal 离子通道传感器不同性质的多种刺激构变化;这些动态构象是挑战, 以确定和仍然很大的未知。近年来, 单粒子低温电子显微镜 (低温 EM) 脱落光对激动剂结合部位的结构特征和几种离子通道的活化机理进行了研究, 确定了其浇口深度动态分析的阶段。使用光谱学方法的机制。电子顺磁共振 (EPR) 和双电子-电子共振 (鹿) 等光谱技术主要局限于可大量纯化的原核离子通道的研究。对大量功能性和稳定的膜蛋白的要求, 阻碍了使用这些方法研究哺乳动物离子通道。EPR 和鹿提供了许多好处, 包括确定移动蛋白区域的结构和动态变化, 虽然在低分辨率, 可能难以获得 X 射线结晶学或低温 EM, 并监测可逆门过渡 (即闭合、开放、敏感和麻木)。在这里, 我们提供的协议, 获取毫克的功能洗涤剂-可溶性瞬态受体电位阳离子通道亚科 V 成员 1 (TRPV1), 可以标记为 EPR 和鹿光谱学。

最小半胱氨酸-少 TRPV1 构造 (eTRPV1) 和单半胱氨酸突变体的功能表征
向光谱学研究迈出的第一步是对功能化和产生生化量蛋白质的半胱氨酸蛋白质结构 (图 2A) 进行设计和表征。eTRPV1 的功能, 由 Ca2 +成像和 TEVC (图 2B-C) 确定。此外, eTRPV1 为 EPR 和鹿实验提供了足够数?...

Watch this Scientific Journal Video about Purification and Reconstitution of TRPV1 for Spectroscopic Analysis at JoVE.com

Purification and Reconstitution of TRPV1 for Spectroscopic Analysis

光谱分析 TRPV1 的纯化与重构

本文介绍了获得洗涤剂-可溶性 TRPV1 的生化量的具体方法, 用于光谱分析。这些联合协议提供了生物化学和生物物理工具, 可用于促进膜控制环境中哺乳动物离子通道的结构和功能研究。

Polymodal ion channels transduce multiple stimuli of different natures into allosteric changes; these dynamic conformations are challenging to determine ...

Research

JoVE Journal

Biochemistry

Method for Efficient Refolding and Purification of Chemoreceptor Ligand Binding Domain

化学感受器配体结合域的高效复用与纯化方法

Identification of natural ligands of chemoreceptors and structural studies aimed at elucidation of the molecular basis of the ligand specificity can be ...

科研

生物化学

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

2在 1: 一步亲和纯化为蛋白质蛋白质和蛋白质代谢物的平行的分析

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Benchtop Immobilized Metal Affinity Chromatography, Reconstitution and Assay of a Polyhistidine Tagged Metalloenzyme for the Undergraduate Laboratory

台式固定化金属亲和层析、Polyhistidine 标记金属酶的重组与检测

Benchtop immobilized metal affinity chromatography (IMAC), of polyhistidine tagged proteins is easily mastered by undergraduate students and has become ...

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

In Vitro Reconstitution of Self-Organizing Protein Patterns on Supported Lipid Bilayers

体外支持性脂质双层自组织蛋白模式的重组

Many aspects of the fundamental spatiotemporal organization of cells are governed by reaction-diffusion type systems. In vitro reconstitution of such ...

Anaerobic Protein Purification and Kinetic Analysis via Oxygen Electrode for Studying DesB Dioxygenase Activity and Inhibition

氧电极对 DesB 酶活性和抑制作用的厌氧蛋白纯化及动力学分析

Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic ...

Expression, Purification, Crystallization, and Enzyme Assays of Fumarylacetoacetate Hydrolase Domain-Containing Proteins

富玛莱酸氢酶含含蛋白质的表达、纯化、结晶和酶测定

Fumarylacetoacetate hydrolase (FAH) domain-containing proteins (FAHD) are identified members of the FAH superfamily in eukaryotes. Enzymes of this ...

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

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

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 ...

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

Isolation of Active Caenorhabditis elegans Nuclear Extract and Reconstitution for In Vitro Transcription

活性 秀丽隐杆线 虫核提取物的分离及重建用于 体外 转录

Caenorhabditis elegans has been an important model system for biological research since it was introduced in 1963. However, C. elegans has not been fully ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

用完全纯化的组分重构Msp1提取活性

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...