Name: site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels
Uploaded: 2016-07-04T15:00:00
Description: Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

我们非常感谢Chakrapani实验室对稿件批判性阅读和评论的现任和前任成员。 这项工作是由卫生部授予全国学院（1R01GM108921）和美国心脏协会（NCRP科学家发展赠款12SDG12070069）和SC的支持。

1.定点突变和Cys突变<ol><li>克隆和诱变 注：GLIC野生型（wt）35有一个单一的天然半胱氨酸（C27），其被突变为丝氨酸以创建一个半胱氨酸较少的背景。半胱氨酸突变引入上通过使用在所需的位置36携带一个半胱氨酸密码子的引物位点定向诱变的半胱氨酸的较少的背景。 <ol><li>混合5微升10×反应缓冲液，1微升100纳克/微升半胱氨酸少GLIC模板DNA 35中 ，每一个40微米的...

EPR谱已被证明是在一个近乎完美的环境量化膜蛋白的构象变化无与伦比的结构方法。这种方法可以让我们一窥了在高分辨率结构遮蔽X射线晶体学和低温电子显微镜蛋白质动力学的分子机制。但是，要考虑这种做法可能会影响一般适用于其他系统，并要牢记沿途的潜在实验路障的技术限制，是非常重要的。我们讨论一些这些方面的下面一起排除故障推荐的策略。 其中较常见的...

在过去的十年，五聚配体门控离子通道的结构理解（pLGIC）增长的跨越式发展，由于家庭的几名成员的高分辨率结构的众人。导致在该领域的当前进展的关键因素包括，原核pLGIC渠道发现，真核生物膜蛋白表达1-3主要进展，4-6和巨大的突破，结构的测定方法。7这些结构上提供了一个明确的共识pLGIC的三维结构的整体保护。然而，这似乎落后背后两大领域是这些渠道制剂的功能特性和通道功能的机械描述。 选通的构象变化是复杂的，发生在沿着通道的长度的60埃的距离和这些转换由广泛调制膜脂。特别是，负脂质，胆固醇和磷脂已经显示调节pLGIC 8-11的功能。尽管通道功能，这些脂质成分的确切作用尚不清楚，门完整的分子机制，需要在他们自然环境研究这些通道。定点自旋标记（SDSL）和电子顺磁共振（EPR）谱是首选在复原系统研究蛋白质动力学的技术。 EPR谱不被分子大小的限制（这是NMR）或样品的光学特性（如为荧光光谱法），由此允许在天然脂质条件重构全长构建体的测量。该技术是非常敏感的，并具有相对低的样品的要求（在微微摩尔的范围）。这两个方面使该技术非常适合于大型研究膜蛋白难以超过毫克来表达数量。 利用与定点自旋标记组合EPR谱是由韦恩哈贝尔和同事开发，并已被改编为研究的范围内的蛋白质的类型。12-24 EPR数据的已被用于研究的二级结构，在该蛋白质改变构象，膜 - 插入深度，以及蛋白质 - 蛋白质/蛋白质 - 配体相互作用。 该方法涉及在由定点诱变感兴趣位置半胱氨酸取代。为确保位点特异性标记，就必须以替代天然半胱氨酸与另一个氨基酸（ 例如 ，丝氨酸）来创建一个半胱氨酸少模板。到目前为止，最流行的自旋标记是一个特定硫醇MTSL：（1-氧基-2,2,5,5-四甲基Δ3吡咯啉-3-甲基）methanethiosulfonate通过二硫键桥连接到蛋白质上。由于其高特异性，相对小的尺寸（小于色氨酸稍大），和灵活接头区域的相容性，这自旋标记已被证明具有优异的反应性，即使埋入半胱氨酸。此外，为了最大限度地提高反应性，该蛋白质的标记反应是在洗涤剂溶解的形式进行。通过尺寸排阻色谱的过量的游离自旋标记的分离后，将蛋白质重组到脂质体或限定的脂质组合物的双层模仿系统。在一般情况下，半胱氨酸诱变耐受良好的蛋白质的大部分地区，和自旋探头的相对小的尺寸会导致极小扰动的二级和三级结构。以确保该变形保留野生型功能，标记和重构通道可以通过膜片钳测量来研究。 然后将标记的官能蛋白进行光谱测量，其基本上提供三种主要类型的信息：由linesh 12,14,15,20,22,23,25-27自旋探针动力学猿分析;探头顺松弛剂无障碍;和距离的分布。27 EPR距离通过两种不同的方法进行测定。第一种是基于所述连续波（CW）技术，其中，从自旋标签之间偶极相互作用产生的频谱展宽（在8 - 20埃的距离范围内）。用于确定距离28,29的第二个是一个脉冲的EPR方法，其中，距离测量可以延长至70埃。30-34在双电子电子共振（鹿），在自旋回波振幅振荡被分析以确定的距离和距离的分布。这里自旋回波是在偶极相互作用的频率调制。总之，这些参数被用来确定蛋白质的拓扑结构，二级结构元件，和蛋白质的构象变化。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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		<tr>
			<td>Site-Directed Mutagenesis and Cys mutations</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10x PfuUltra HF reaction buffer</td>
			<td>Agilent Technologies</td>
			<td>600380-52</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPS</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>N0447L</td>
			<td>10mM each dNTP</td>
		</tr>
		<tr>
			<td>pfu Ultra DNA polymerase</td>
			<td>Agilent Technologies</td>
			<td>600380-51</td>
			<td>2.5 U/ul</td>
		</tr>
		<tr>
			<td>DPNI</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>R0176S</td>
			<td>20,000 U/ml</td>
		</tr>
		<tr>
			<td>XL10 GOLD</td>
			<td>Agilent Technologies</td>
			<td>200314</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC media</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>B9020S</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Fisher Scientfic</td>
			<td>BP905</td>
			<td></td>
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		<tr>
			<td>LB media</td>
			<td>Invitrogen</td>
			<td>127957084</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>QIAGEN</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>C43 competent cells</td>
			<td>Lucigen</td>
			<td>60446</td>
			<td></td>
		</tr>
		<tr>
			<td>Expression and Purification</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Fisher Scientfic</td>
			<td>D16</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>Fisher Bioreagents</td>
			<td>BP1421-500</td>
			<td></td>
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			<td>Yeast extract</td>
			<td>Amresco</td>
			<td>J850</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Bioreagents</td>
			<td>BP229</td>
			<td></td>
		</tr>
		<tr>
			<td>K2HPO4</td>
			<td>Amresco</td>
			<td>0705</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Amresco</td>
			<td>0781</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG (isopropyl-thio-&#946;-galactoside)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C25</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma Life Science</td>
			<td>T1503</td>
			<td></td>
		</tr>
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			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>Sigma Life Science</td>
			<td>DN25</td>
			<td></td>
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		<tr>
			<td>PMSF</td>
			<td>Amresco</td>
			<td>M145</td>
			<td></td>
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		<tr>
			<td>Leupeptine</td>
			<td>Amresco</td>
			<td>J580</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepstatin</td>
			<td>Amresco</td>
			<td>J583</td>
			<td></td>
		</tr>
		<tr>
			<td>DDM (n-Docecyl-&#946;-D-Maltopyranoside)</td>
			<td>Anatrace</td>
			<td>D310S</td>
			<td></td>
		</tr>
		<tr>
			<td>Amylose resin</td>
			<td>New England BioLabs Inc&#8206;</td>
			<td>E8021L</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP</td>
			<td>Amresco</td>
			<td>K831</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientfic</td>
			<td>BP118</td>
			<td></td>
		</tr>
		<tr>
			<td>Maltose</td>
			<td>Acros Organics</td>
			<td>329915000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superdex 200GL</td>
			<td>GE Healthcare</td>
			<td>17-5175-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Empty polypropylene Chromatography column</td>
			<td>BioRad</td>
			<td>731-1550</td>
			<td></td>
		</tr>
		<tr>
			<td>Site-Directed Spin Labeling</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MTSL (1-oxyl-2,2,5,5-tetramethyl-3-pyrroline-3-methyl) Methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>O873900</td>
			<td></td>
		</tr>
		<tr>
			<td>(1-acetoxy-2,2,5,5-tetramethyl-&#916;3-pyrroline-3-methyl) methanethiosulfonate</td>
			<td>Toronto Reaserch chemicals Inc</td>
			<td>A167900</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>J.T. Baker</td>
			<td>9224-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Reconstitution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Asolectin lipid</td>
			<td>Avanti polar lipids Inc</td>
			<td>541602C</td>
			<td></td>
		</tr>
		<tr>
			<td>Biobeads (Polystyrine beads)</td>
			<td>Bio Rad</td>
			<td>152-3920</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher chemicals</td>
			<td>A413</td>
			<td></td>
		</tr>
		<tr>
			<td>FRET</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>F-150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylrhodamine-maleimide</td>
			<td>ThermoFisher Scientific</td>
			<td>T-6027</td>
			<td></td>
		</tr>
		<tr>
			<td>POPC</td>
			<td>Avanti polar lipids Inc</td>
			<td>850457C</td>
			<td></td>
		</tr>
		<tr>
			<td>POPG</td>
			<td>Avanti polar lipids Inc</td>
			<td>840457C</td>
			<td></td>
		</tr>
		<tr>
			<td>E.Coli polar lipid extract</td>
			<td>Avanti polar lipids Inc</td>
			<td>100600C</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma Life Science</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr>
			<td>EPR measurement</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TPX plastic capillaries</td>
			<td>Bruker</td>
			<td>ER221</td>
			<td></td>
		</tr>
		<tr>
			<td>EDDA (Ethylenediamine-N, N&#39;-diacetic acid)</td>
			<td>Aldrich</td>
			<td>158186</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni(OH)2</td>
			<td>Aldrich</td>
			<td>283622</td>
			<td></td>
		</tr>
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</table>

site directed spin labeling and epr spectroscopic studies of pentameric ligand-gated ion channels

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. Fundamental to these transitions is the intrinsic flexibility of the protein, which is critically modulated by membrane lipid-composition. To better understand the structural basis of channel function, it is necessary to study protein dynamics in a physiological membrane environment. Electron Paramagnetic Resonance (EPR) spectroscopy is an important tool to characterize conformational transitions between functional states. In comparison to NMR and X-ray crystallography, the information obtained from EPR is intrinsically of lower resolution. However, unlike in other techniques, in EPR there is no upper-limit to the molecular weight of the protein, the sample requirements are significantly lower, and more importantly the protein is not constrained by the crystal lattice forces. Therefore, EPR is uniquely suited for studying large protein complexes and proteins in reconstituted systems. In this article, we will discuss general protocols for site-directed spin labeling and membrane reconstitution using a prokaryotic proton-gated pentameric Ligand-Gated Ion Channel (pLGIC) from Gloeobacter violaceus (GLIC) as an example. A combination of steady-state Continuous Wave (CW) and Pulsed (Double Electron Electron Resonance-DEER) EPR approaches will be described that will enable a complete quantitative characterization of channel dynamics.

自旋标记GLIC突变体的生化特征 按照上述描述的方案通常产生GLIC-MBP融合蛋白在10〜 - 12毫克/升培养物。虽然这个值可以在不同的突变体而变化，特别是对于埋在蛋白质内的位置时，产率可能会显著损害。在这些情况下，培养体积可能需要扩大。融合的裂解构建由HRV3C蛋白酶进行O / N方便。通过运行在SDS PAGE凝胶上?...

Watch this Scientific Journal Video about Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels at JoVE.com

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

定点自旋标记和五聚体配体门控离子通道的EPR光谱研究

Ion channel gating is a stimulus-driven orchestration of protein motions that leads to transitions between closed, open, and desensitized states. ...

Research

JoVE Journal

Chemistry

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

小型低聚物形成膜蛋白的沉降平衡：组氨酸质子对五聚体稳定性

Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and ...

科研

化学

Transport of Surface-modified Carbon Nanotubes through a Soil Column

表面修饰碳纳米管通过土柱运输

Carbon nanotubes (CNTs) are widely manufactured nanoparticles, which are being utilized in a number of consumer products, such as sporting goods, ...

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

弥漫分散剂/粒子绑定机制反射红外光谱鉴定功能性油墨

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers.  A typical inkjet ink is a colloidal ...

A Filter-based Surface Enhanced Raman Spectroscopic Assay for Rapid Detection of Chemical Contaminants

基于过滤器的表面增强拉曼光谱检测方法的化学污染物快速检测

We demonstrate a method to fabricate highly sensitive surface-enhanced Raman spectroscopic (SERS) substrates using a filter syringe system that can be ...

Using Cyclic Voltammetry, UV-Vis-NIR, and EPR Spectroelectrochemistry to Analyze Organic Compounds

利用循环伏安法、紫外-近红外光谱和 EPR 光谱电化学对有机化合物进行分析

Cyclic voltammetry (CV) is a technique used in the analysis of organic compounds. When this technique is combined with electron paramagnetic resonance ...

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

Syntheses, Crystallization, and Spectroscopic Characterization of 3,5-Lutidine N-Oxide Dehydrate

35-吡啶N-氧化物脱水的合成、结晶和光谱表征

The synthesis of 3,5-lutidine N-oxide dehydrate, 1, was achieved in the synthesis route of 2-amino-pyridine-3,5-dicarboxylic acid. Ochiai first used the ...

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

局部钙离子梯度对大泡囊膜重塑的响应

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

用于测定金属结合寡聚肽的金属电子识别和机制的Ion流动性-质谱技术

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

信息承载Peptoids及其序列定向动态共价自组装的合成

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

将类似蛋白的蛋白质定向组装到定义的超分子结构和体外货物封装中

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery ...