Name: voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids
Uploaded: 2017-05-27T13:00:00
Description: Watch this Scientific Journal Video about Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids at JoVE.com

pAnap是Peter Schultz博士（斯克里普斯研究所）的礼物。这项工作由加拿大卫生研究所MOP-102689和MOP-136894（加拿大RB）和加拿大创新基金会950-225005资助。

青蛙操作按照加拿大指南进行，并经蒙特利尔大学道德委员会（CDEA，协议＃15-042）的批准。 1. fUAA公司的mRNA准备<ol><li>选择蛋白质感兴趣的位置，预期会发生构象变化。选择该区域中的氨基酸代替fUAA。 注意：位置的选择是基于预期的结构重新排列。如果存在高分辨率结构和预期运动的假设，则应该放置该化学物质的环境将会改变;这可能是介电常数（疏水性与亲水性环...

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与tRNA合成酶连续转录的tRNA 的体内氨基酰化使得可以获得荧光测量的高表达水平。为了有效的fUAA并入，pAnap正确注入细胞核是至关重要的。由于核的确切位置的不确定性，预计10-40％的DNA注射会失败，导致不表达（或泄漏表达）卵母细胞。因此，在没有Anap和pAnap的情况下检查表达是重要的，以识别泄漏通道情况下的当前表型和幅度。这样，通过适当的DNA注射（高表达，Anap dF，全长当前表型?...

具有各种化学和物理性质的200多种非天然氨基酸被遗传并入大肠杆菌 ，酵母和哺乳动物细胞中的蛋白质3 。非天然氨基酸通过正交工程化的tRNA /合成酶对而与特异性终止密码子相结合。改变蛋白质的遗传学方法为蛋白质结构和功能提供了宝贵的见解。在这里，我们提出使用电压钳夹荧光测量（VCF）与荧光UAA组合的方案。 在VCF中，同时观察位于荧光探针周围的功能数据和结构重排（〜5Å）可使我们以毫秒级分辨率获得动态信息1 。荧光探针在蛋白质定位运动时改变其淬灭状态。只有1-2埃的运动足以导致荧光的显着变化强度4 。鉴定目标蛋白质感兴趣位点后，通过点突变突变位点。经典地，残基已被突变为半胱氨酸，而现在，引入琥珀终止密码子（TAG）用于遗传fUAA并入。然后蛋白质被体外转录。 虽然其他表达系统（ 例如哺乳动物细胞）可以使用5,6,7 ， 非洲爪蟾卵母细胞因结构功能研究而较大，因为其尺寸较大，导致操作更容易，荧光强度更高（更多的荧光团），因此，信噪比。此外， 非洲爪蟾卵母细胞具有低内源性蛋白质的背景2,8，动物杆上的黑色色素沉着抵抗来自t的背景荧光他的细胞质手术切除非洲爪蟾卵母细胞，将编码fUAA特异性的正交tRNA / tRNA-合成酶对的DNA注射到卵母细胞核中。在孵育6-24小时后，将蛋白质RNA与fUAA共注射到卵母细胞的细胞溶质中，随后孵育2-3天。为了防止对fUAA（光漂白）的任何损坏，包括Anap在内的程序必须在红光下进行，以避免荧光团激发。 在切开的卵母细胞电压钳设置上研究卵母细胞，其安装在直立荧光显微镜上，电流和荧光变化同时记录9,10 。或者，可以使用双电极电压钳1或膜片钳构造11 。荧光被具有低RMS噪声的适当波长激发<html>使用连接到具有高放大率的放大器的光电二极管记录的发射。 在电压钳荧光测定中使用荧光非天然氨基酸（fUAAs）有几个优点。一个是进入膜蛋白质的胞质侧;许多调节过程位于这里（ 例如， Ca 2+ - 或核苷酸结合位点，电压门控离子通道的快速和闭态失活，开孔，模块耦合）。所有这些过程现在都可用于荧光标记。 另一个优点是探针的小尺寸导致蛋白质的较少干扰。到目前为止，fUAAs的两个正交tRNA / tRNA合成酶对已被设计为12,13 ，其中3-（6-乙酰基萘-2-基氨基）-2-氨基丙酸（Anap）是唯一已被用于非洲爪蟾卵母细胞的fUAA 如图2所示 ，“xref”&gt; 8。 Anap是一种环境敏感的荧光团，分子量为272.3 g / mol，仅略高于色氨酸12 （ 图1A，1B ）。由于其小的尺寸，与通过接头连接的常规荧光团（通常大于500g / mol）相比，荧光团可能引入较少的空间效应。此外，在Anap的情况下，荧光团比与半胱氨酸相连的位置更靠近蛋白质主链，因此，Anap正在探测更局部的重排。最后，在UAA-VCF中不再需要在常规VCF中去除内源性半胱氨酸以确保位点特异性标记，因此（i）使蛋白质（几乎）为其天然状态，（ii）允许VCF应用研究更广泛的蛋白质，其功能可能被半胱氨酸取代改变。 <img alt="图1" src =“/ files / ftp_upload / 55598 / 55598fig1.jpg”/&gt; 图1 ：Anap和荧光光谱。 （ A ）Anap的化学结构。 （ B ）1nM Anap的归一化吸收光谱和发射光谱，证明Anap荧光对溶剂疏水性的敏感性。通过在350nm激发获得发射光谱。 <a href="https://www.jove.com/files/ftp_upload/55598/55598fig1large.jpg" target="_blank">请点击此处查看此图的较大版本。</a> 使用荧光UAA的缺点是如果氨基酰化tRNA的量稀少，则异源蛋白质群体可能由终止密码子读取，翻译重新启动，C-末端截短的蛋白质或内源氨基酰化的串扰引起。在不存在fUAA和tRNA / tRNA合成酶对的情况下，应始终检查这种泄漏表达。我们处理了转交的问题国际重新启动以及如何规避N末端插入位置以前14 。然而，当fUAA，tRNA和tRNA合成酶以饱和量存在时，仅存在泄漏表达的可能性较低。 fUAA-VCF与常规VCF的关键程序差异在于注射和处理卵母细胞;注射编码tRNA和tRNA合成酶（pAnap）的DNA之后，引入Anap，其与蛋白质mRNA共注射，或者作为乙酰氧基甲基（AM）酯加入到孵育溶液中。

<table border="0" cellpadding="0" cellspacing="0" width="567">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;">Solutions</td>
			<td style="width:111px;"></td>
			<td style="width:161px;"></td>
			<td style="width:96px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Barth&#39;s solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>90mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Fisher Scientific</td>
			<td>BP366-500</td>
			<td>3mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>M-9397</td>
			<td>0.82mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C-7902</td>
			<td>0.41mM</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Ca(NO3)2</td>
			<td>Sigma-Aldrich</td>
			<td>C-1396</td>
			<td>0.33mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaOH hydrate</td>
			<td>BDH</td>
			<td>BDH7225-4</td>
			<td>pH 7.6</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Penicilin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100U/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Streptomycin</td>
			<td>Invitrogen</td>
			<td>15140122</td>
			<td>100&#181;g/mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kanamycin</td>
			<td>Invitrogen</td>
			<td>15160054</td>
			<td>10mg/100mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Horse serum</td>
			<td>Invitrogen</td>
			<td>16050122</td>
			<td>5%</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SOS Standard Oocyte Solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NaCl&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>746398</td>
			<td>102 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KCl</td>
			<td>Sigma-Aldrich</td>
			<td>746436</td>
			<td>3 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M9272</td>
			<td>1 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>5 mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">External recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Internal recording solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">N-methyl-D-glucamine (NMDG)</td>
			<td>Alfa Aesar</td>
			<td>L14282</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethylenediamine Tetraacetic Acid (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>E478-500</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Labeling solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KOH</td>
			<td>Fisher Scientific</td>
			<td>P250-1</td>
			<td>115mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H4034</td>
			<td>10mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Calcium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>239232</td>
			<td>2mM</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MES hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>258105</td>
			<td>pH 7.2</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMR stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tetramethylrhodamine-5-maleimide (TMR)</td>
			<td>Molcular Probes by Life Technologies</td>
			<td>T6027</td>
			<td>5mM in DMSO</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anap stock solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">Anap</td>
			<td>ABZENA (TCRS)</td>
			<td>Custom synthesis TCRS-170</td>
			<td style="width:96px;">1mM in nuclease-free water and 1% NaOH 1N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Material/Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">pAnap</td>
			<td>Addgene</td>
			<td>48696</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Performance Oocyte Clamp</td>
			<td>Dagan Corporation</td>
			<td>CA-1B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gpatch Acquisition software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Analysis software</td>
			<td colspan="3">Department of Anesthesiology, University of California, Los Angeles</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Recording Chamber</td>
			<td colspan="2">Custom machined</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photo diode detection system</td>
			<td>Dagan Corporation</td>
			<td>PhotoMax-200/PIN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electrical shutter driver</td>
			<td>UNIBLITZ</td>
			<td>VCM-D1</td>
			<td></td>
		</tr>
	</tbody>
</table>

voltage-clamp fluorometry in xenopus oocytes using fluorescent unnatural amino acids

电压钳式荧光法（VCF）一直是研究电生物膜蛋白的结构和功能的选择技术，其中荧光和电流的实时测量同时报告局部重排和全局功能。虽然高分辨率结构技术如低温电子显微镜或X射线晶体学提供了感兴趣的蛋白质的静态图像，但VCF提供了动态结构数据，使我们能够将结构重排（荧光）与动态功能数据（电生理学）联系起来。直到最近，用于蛋白质定点荧光标记的硫醇反应性化学物质限制了该方法的范围，因为所有可及的半胱氨酸，包括内源性半胱氨酸都将被标记。因此需要构建不含内源性半胱氨酸的蛋白质。标记也限于可从细胞外获得的位点侧。随着使用荧光非天然氨基酸 （fUAA），使用正交tRNA和tRNA合成酶对2特异性并入小荧光探针以响应终止密码子抑制而改变。 VCF改进仅需要DNA注射（tRNA /合成酶对）的两步注射程序，然后进行RNA / fUAA共注射。现在，标记细胞内和掩埋的位点是可能的，VCF的使用已经显着扩大。因此，VCF技术对于研究广泛的蛋白质变得有吸引力，更重要的是允许研究许多细胞溶质调节机制。

图4显示了在存在pAnap和Anap的情况下，从表达快速灭活快速灭活的卵母细胞（IR），L382stop-W434F的卵母细胞获得的VCF记录的实例。 W434F突变阻断离子钾电流，这使得可以测量瞬态门控电荷位移（门电流）。门极电流（上限曲线）和Anap荧光强度变化（下限轨迹）同时记录去极化表明Anap成功纳入了位置L382。在这里，Anap位于在激活期间移动的四个S4跨膜螺旋中的每?...

Watch this Scientific Journal Video about Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids at JoVE.com

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

本文介绍了使用荧光非天然氨基酸（fUAA）代替马来酰亚胺染料的常规电压钳子荧光测量（VCF）的增强，以探测离子通道中的结构重排。该程序包括非洲爪蟾卵母细胞DNA注射，RNA / fUAA共注射，以及同步电流和荧光测量。

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Research

JoVE Journal

Biochemistry

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

A Polyaniline-based Sensor of Nucleic Acids

核酸的聚苯胺传感器

Detection of nucleic acids is at the center of diagnostic technologies used in research and the clinic. Standard approaches used in these technologies ...

科研

生物化学

An Optimized Protocol for Electrophoretic Mobility Shift Assay Using Infrared Fluorescent Dye-labeled Oligonucleotides

一种优化的协议电泳迁移分析使用红外荧光染料标记的寡核苷酸

Electrophoretic Mobility Shift Assays (EMSA) are an instrumental tool to characterize the interactions between proteins and their target DNA sequences. ...

Identification of Fatty Acids in Bacillus cereus

Identification of Fatty Acids in Bacillus cereus

脂肪酸鉴定<em&gt;蜡状芽孢杆菌</em

The Bacillus species contain branched chain and unsaturated fatty acids (FAs) with diverse positions of the methyl branch (iso or anteiso) and of the ...

DNA Polymerase Activity Assay Using Near-infrared Fluorescent Labeled DNA Visualized by Acrylamide Gel Electrophoresis

用丙烯酰胺凝胶电泳法可视化近红外荧光标记 dna 聚合酶活性

For any enzyme, robust, quantitative methods are required for characterization of both native and engineered enzymes. For DNA polymerases, DNA synthesis ...

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

Reconstitution of Cell-cycle Oscillations in Microemulsions of Cell-free Xenopus Egg Extracts

无细胞爪卵萃取物微乳液细胞周期振荡的重建

Real-time measurement of oscillations at the single-cell level is important to uncover the mechanisms of biological clocks. Although bulk extracts ...

Structure Solution of the Fluorescent Protein Cerulean Using MeshAndCollect

利用网格和收集荧光蛋白陶瓷的结构解

X-ray crystallography is the major technique used to obtain high resolution information concerning the 3-dimensional structures of biological ...

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

聚丙烯酰胺凝胶中蛋白质的荧光银染色

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Identifying Amino Acid Overproducers Using Rare-Codon-Rich Markers

使用稀有的科顿富标记识别氨基酸过度生产者

To satisfy the ever-growing market for amino acids, high-performance production strains are needed. The amino acid overproducers are conventionally ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

荧光苏门答腊蛋白对 sumo 修饰蛋白的定位

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...