Name: monitoring gpcr-&#946;-arrestin1/2 interactions in real time living systems to accelerate drug discovery
Uploaded: 2019-06-28T14:15:00
Description: Watch this Scientific Journal Video about Monitoring GPCR-ÃŽÂ²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery at JoVE.com

这项工作得到了韩国国家研究基金会(NRF)(NRF-2015M3A9E7029172)的资助,该研究由科学、ICT和未来规划部资助。

1. 引性设计策略
<ol><li>设计引源,将感兴趣的基因引入 pBiT1.1-C [TK/LgBiT]、pBiT2.1-C [TK/SmBiT]、pBiT1.1-N [TK/LgBiT] 和 pBiT2.1-N [TK/SmBiT] 矢量图。</li>
	<li>选择这三个位点中至少一个作为定向克隆所需的两种唯一限制酶之一,因为存在一个帧内停止通子,该块子将多克隆位点分开,如图11所示。</li>
	<li>如表1所示,将核苷酸序列纳入引物中,以编码表2</strong...

<ol>
	<li>Sriram, K., Insel, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCRs+as+targets+for+approved+drugs:+How+many+targets+and+how+many+drugs?.">GPCRs as targets for approved drugs: How many targets and how many drugs?.</a> Molecular Pharmacology. 93 (4), 251-258 (2018).</li><li>Hauser, A. S., Attwood, M. M., Rask-Andersen, M., Schi&#246;th, H. B., Gloriam, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+GPCR+drug+discovery:+new+agents,+targets+and+indications.">Trends in GPCR drug discovery: new agents, targets and indications.</a> Nature Reviews Drug Discovery. 16 (12), 829-842 (2017).</li><li>Langmead, C. J., Summers, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pharmacology+of+GPCRs.">Molecular pharmacology of GPCRs.</a> British Journal of Pharmacology. 175 (21), 1754005-1754008 (2018).</li><li>Lohse, M. J., Hoffmann, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arrestin+Interactions+with+G+Protein-Coupled+Receptors.">Arrestin Interactions with G Protein-Coupled Receptors.</a> Handbook of Experimental Pharmacology. 219, 15-56 (2014).</li><li>Kang, D. S., 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=Structure+of+an+arrestin2-clathrin+complex+reveals+a+novel+clathrin+binding+domain+that+modulates+receptor+trafficking.">Structure of an arrestin2-clathrin complex reveals a novel clathrin binding domain that modulates receptor trafficking.</a> Journal of Biological Chemistry. 284, 29860-29872 (2009).</li><li>Park, S. 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=Effects+of+&#946;-Arrestin-Biased+Dopamine+D2+Receptor+Ligands+on+Schizophrenia-Like+Behavior+in+Hypoglutamatergic+Mice.">Effects of &#946;-Arrestin-Biased Dopamine D2 Receptor Ligands on Schizophrenia-Like Behavior in Hypoglutamatergic Mice.</a> Neuropsychopharmacology. 41 (3), 704-715 (2016).</li><li>Zhu, L., Cui, Z., Zhu, Q., Zha, X., Xu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Opioid+Receptor+Agonists+with+Reduced+Morphine-like+Side+Effects.">Novel Opioid Receptor Agonists with Reduced Morphine-like Side Effects.</a> Mini-Reviews in Medicinal Chemistry. 18 (19), 1603-1610 (2018).</li><li>Smith, J. S., Lefkowitz, R. J., Rajagopal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+signalling:+from+simple+switches+to+allosteric+microprocessors.">Biased signalling: from simple switches to allosteric microprocessors.</a> Nature Reviews Drug Discovery. 17 (4), 243-260 (2018).</li><li>Dixon, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Reyes-Alcaraz, A., Lee, Y. N., Yun, S., Hwang, J. I., Seong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+signatures+in+&#946;-arrestin2+reveal+natural+biased+agonism+at+a+G-protein-coupled+receptor.">Conformational signatures in &#946;-arrestin2 reveal natural biased agonism at a G-protein-coupled receptor.</a> Communications Biology. 3, 1-128 (2018).</li><li>Promega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nanobit Protein Protein Interaction System Protocol. , (2019).</li><li>Life Biomedical. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> HiYield Gel/PCR Fragments Extraction Kit. , (2019).</li><li>New England BioLabs. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Ligation Calculator. , (2019).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cosmo Genetech. , (2019).</li><li>Baggio, L. L., Drucker, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+incretins:+GLP-1+and+GIP.">Biology of incretins: GLP-1 and GIP.</a> Gastroenterology. 132, 2131-2157 (2007).</li><li>ProMega. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> NanoGLO Endurazine and Vivazine Live Cell Substrates Technical Manual. , (2019).</li><li>Ali, R., Ramadurai, S., Barry, F., Nasheuer, H. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+fluorescent+protein+expression+for+quantitative+fluorescence+microscopy+and+spectroscopy+using+herpes+simplex+thymidine+kinase+promoter+sequences.">Optimizing fluorescent protein expression for quantitative fluorescence microscopy and spectroscopy using herpes simplex thymidine kinase promoter sequences.</a> FEBS Open Bio. 8 (6), 1043-1060 (2018).</li></ol>

使用此处介绍的方法,使用此 GPCR-β-逮捕蛋白结构补充测定,可在实时生命系统中监控任何 GPCR 和 β-阻尼1/2之间的相互作用。在这方面,我们能够观察到GLP-1r(一种典型的B类GPCR)的两种β-逮捕素异种的招募,我们还观察到受体-β-逮捕复合物在达到最大值后几分钟内分离发光信号。
为了在结构补性测定系统中具有最佳灵敏度,在受体和每个β-阻尼素异构体之间对它进行了四种不同的空...

GPCRs代表了市场上近35%的当前药物的目标1、2,而对其药理学的清晰了解对于开发新型治疗药物3至关重要。GPCR药物发现的关键方面之一,特别是在偏置激动剂的发育过程中,是新配体对受体-β-逮捕素相互作用4和β-逮捕蛋白与其他细胞蛋白相互作用的表征,例如作为克拉特林5。
据记载,β-arrestin依赖信号在神经紊乱(如双相情感障碍、严重抑郁症和精神分裂症6)中起关键作用,在一些药物(如吗啡7)中也有严重的副作用。
目前用于监测这些相互作用的方法通常不代表研究中蛋白质的实际内源水平,在某些情况下,它们显示信号弱、光漂白,并且根据GPCR,建立8可能具有技术挑战性。这种新颖的结构补体测定使用低表达促进因子载体来模拟内源性生理水平,与目前的方法9相比,具有较高的灵敏度。使用这种方法,可以容易地描述Galanin受体-β-逮捕1/2和β-逮捕2-克拉林相互作用10。这种方法可以广泛用于任何特别感兴趣的GPCR,其中β-逮捕蛋白发挥关键的生理功能或其信号在某些疾病中相关。

<table>
	<tbody>
		<tr>
			<td>Antibiotics penicillin streptomycin</td>
			<td>Welgene</td>
			<td>LS202-02</td>
			<td>Penicillin/Streptomycin</td>
		</tr>
		<tr>
			<td>Bacterial Incubator</td>
			<td>JEIO Tech</td>
			<td>IB-05G</td>
			<td>Incubator (Air-Jacket), Basic</td>
		</tr>
		<tr>
			<td>Cell culture medium</td>
			<td>Welgene</td>
			<td>LM 001-05</td>
			<td>DMEM Cell culture medium</td>
		</tr>
		<tr>
			<td>Cell culture transfection medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td>Optimem 1X cell culture medium</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>NUAIRE</td>
			<td>NU5720</td>
			<td>Direct Heat CO2 Incubator</td>
		</tr>
		<tr>
			<td>Digital water bath</td>
			<td>Lab Tech</td>
			<td>LWB-122D</td>
			<td>Digital water bath lab tech</td>
		</tr>
		<tr>
			<td>DNA Polymerase proof reading</td>
			<td>ELPIS Biotech</td>
			<td>EBT-1011</td>
			<td>PfU DNA polymerase</td>
		</tr>
		<tr>
			<td>DNA purification kit</td>
			<td>Cosmogenetech</td>
			<td>CMP0112</td>
			<td>miniprepLaboPass Purificartion Kit Plasmid Mini</td>
		</tr>
		<tr>
			<td>DNA Taq Polymerase</td>
			<td>Enzynomics</td>
			<td>P750</td>
			<td>nTaq DNA polymerase</td>
		</tr>
		<tr>
			<td>Enzyme restriction BglII</td>
			<td>New England Biolabs</td>
			<td>R0144L</td>
			<td>BglII</td>
		</tr>
		<tr>
			<td>Enzyme restriction buffer</td>
			<td>New England Biolabs</td>
			<td>B72045</td>
			<td>CutSmart 10X Buffer</td>
		</tr>
		<tr>
			<td>Enzyme restriction EcoRI</td>
			<td>New England Biolabs</td>
			<td>R3101L</td>
			<td>EcoRI-HF</td>
		</tr>
		<tr>
			<td>Enzyme restriction NheI</td>
			<td>New England Biolabs</td>
			<td>R01315</td>
			<td>NheI</td>
		</tr>
		<tr>
			<td>Enzyme restriction XhoI</td>
			<td>New England Biolabs</td>
			<td>R0146L</td>
			<td>XhoI</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Gibco Canada</td>
			<td>12483020</td>
			<td>Fetal Bovine Serum</td>
		</tr>
		<tr>
			<td>Gel/PCR DNA MiniKit</td>
			<td>Real Biotech Corporation</td>
			<td>KH23108</td>
			<td>HiYield Gel/PCR DNA MiniKit</td>
		</tr>
		<tr>
			<td>Ligase</td>
			<td>ELPIS Biotech</td>
			<td>EBT-1025</td>
			<td>T4 DNA Ligase</td>
		</tr>
		<tr>
			<td>Light microscope</td>
			<td>Olympus</td>
			<td>CKX53SF</td>
			<td>CKX53 Microscope Olympus</td>
		</tr>
		<tr>
			<td>lipid transfection reagent</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Lipofectamine 2000</td>
		</tr>
		<tr>
			<td>Luminometer</td>
			<td>Biotek/Fisher Scientific</td>
			<td>12504386</td>
			<td>Synergy 2 Multi-Mode Microplate Readers</td>
		</tr>
		<tr>
			<td>NanoBiT System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>NanoBiT PPI MCS Starter System</td>
		</tr>
		<tr>
			<td>Nanoluciferase substrate</td>
			<td>Promega</td>
			<td>N2012</td>
			<td>Nano-Glo Live Cell assay system</td>
		</tr>
		<tr>
			<td>PCR Thermal cycler</td>
			<td>Eppendorf</td>
			<td>6336000015</td>
			<td>Master cycler Nexus SX1</td>
		</tr>
		<tr>
			<td>Poly-L-lysine</td>
			<td>Sigma Aldrich</td>
			<td>P4707-50ML</td>
			<td>Poly-L-lysine solution</td>
		</tr>
		<tr>
			<td>Trypsin EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td>Trysin EDTA 10X</td>
		</tr>
		<tr>
			<td>White Cell culture 96 well plates</td>
			<td>Corning</td>
			<td>3917</td>
			<td>Assay Plate 96 well plate</td>
		</tr>
	</tbody>
</table>

monitoring gpcr-&#946;-arrestin1/2 interactions in real time living systems to accelerate drug discovery

G-蛋白耦合受体(GPCRs)和β-失能素之间的相互作用是具有非常重要生理影响的重要过程。目前,在GPCR药物发现领域,特别是在GPCR偏置症研究期间,新药物的表征与β-逮捕蛋白和其他细胞蛋白的相互作用具有极其宝贵的价值。在这里,我们展示了一种新型结构补体测定的应用,以准确监测实时生命系统中的受体-β-逮捕素相互作用。该方法简单、准确,易于扩展到任何感兴趣的GPCR,而且它的优点是克服了由于每个向量系统中存在低表达启动子而导致的非特定交互。这种结构补加测定提供了关键特性,能够准确、准确地监测受体-β-失素相互作用,使其适用于研究任何GPCR系统的偏置性痛苦以及GPCR c-终点的磷酸化由不同的GPCR-激酶(GRKs)和后翻译修改的arrestins,稳定或稳定受体-β-逮捕复合物的代码。

使用此处介绍的程序,监测了典型GPCR和两种β-逮捕蛋白异体之间的相互作用。胰高血糖素类似肽受体(GLP-1r)结构是使用含有NheI和EcoRI酶限制性位点的引基体,并克隆到载体pBiT1.1-C[TK/LgBiT]和pBiT2.1-C[TK/SmBiT],而在β-阻尼的情况下,另外两个载体是在β-逮捕2和NheI和XhoI的情况下使用pBiT1-N[TK/LgBiT]和pBiT2.1-N[TK/SmBiT]在β-逮捕1的情况下使用酶抑制位BgIII和EcoRI。HEK293细胞在N-或C端接端使用GLP-1r-LgBiT/SmBiT和50 ng的β-阻...

Watch this Scientific Journal Video about Monitoring GPCR-ÃŽÂ²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery at JoVE.com

Monitoring GPCR-ÃƒÅ½Ã‚Â²-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

GPCR-β-阻尼相互作用是GPCR药物发现中的一个新兴领域。精确、精确和易于设置的方法对于监控生活系统中的这种相互作用是必要的。我们展示了一种结构补体测定,以监测实时活细胞中的GPCR-β-阻尼蛋白相互作用,并可扩展到任何GPCR。

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. ...

该协议在药物发现和开发领域具有很高的影响，特别是在开发具有最小副作用的新颖、一流的药物方面。该技术的主要优点是可应用于任何GPCR系统，并用于获取实时生活系统中受体药理学的准确信息。这项技术的含义与新疗法密切相关，特别是在β-卡贝因相关方面。这些包括疼痛中的蛋白石受体和精神分裂症中的多巴胺受体。该方法在神经病、心肺病等许多医学科学研究领域都很有用。PCR 演示我们的方法非常重要，因为它允许详细介绍协议中的关键步骤，而传统发布则无法实现这一点。首先设计底图，将感兴趣的基因引入pBIT向量。由于划分多克隆位点的 inframe stop codon，因此在三个站点中选择至少一个作为定向克隆所需的两种唯一限制酶之一。有关核苷酸序列的手稿，请对链接器残留物进行编码，并将其合并到底原上。对于 pBIT1.1-C 和 pBIT2.1-C，确保正向底转包含 ATG codon 和强大的 Kozak 共识序列。对于 pBIT1.1-N 和 pBIT2.1-N，请确保反向底法包含止损胶。使用设计底图放大感兴趣的基因的插入DNA。根据手稿方向组合 PCR 试剂，确保使用高保真 DNA 聚合酶以最大限度地减少突变。对热循环器进行编程并开始反应。完成PCR后，在50微升消化反应中消化产品。在1.5毫升管中，组合12微升蒸馏水、5微升适当的10X限制消化缓冲液和30微升PCR产品。然后加入每个限制酶的1.5微升。漩涡反应混合物。并在37.5摄氏度过夜孵育。准备第二反应来消化接受者质粒。在1.5毫升管中，结合23微升水、5微升适当的10X限制消化缓冲液、15微升的受助质粒和1.5微升的每个限制性酶。将管子简单混合。并在37.5摄氏度过夜孵育。在克隆和转化的第二天，选择3到10个单独的细菌菌落，并把它们转移到一毫升的LB培养基与安皮西林。孵育细胞6小时。然后将 200 微升的细菌悬浮液转移到 5 毫升的新鲜 LB 介质中，并使用安霉素。在 37.5 摄氏度下孵育培养，在 200 rpm 下一夜摇晃。第二天，用微型预制备DNA纯化试剂盒分离质粒。筛选纯化质粒，成功使用 PCR 进行连接。转染前一天，使用Dulbecco改良的鹰的介质在涂有96井的聚L-lysine板上播种细胞，并辅以10%的胎儿牛血清、每毫升青霉素G100个单位和每毫升链霉素100微升。仅向 60 个内孔添加细胞，以最大限度地减少整个板的热梯度和蒸发产生的边缘效应。在36口井中加入200微升无菌蒸馏水，在井之间的空间中加入150微升。然后在37.5摄氏度和5%的二氧化碳下孵育板。第二天，使用总共100纳米DNA进行转染。根据手稿方向设置四种不同的质粒组合。使用 20 微升的改性 Eagle 最低基本介质缓冲与 HEPES，以及每井 0.3 微升脂质转染试剂。在每个井中加入20微升脂质转染试剂和DNA混合物，将板在圆中混合10秒。在37.5摄氏度和5%的二氧化碳下孵育6小时。刷新介质。再孵育24小时孵育后，吸气介质，并添加100微升的修改鹰的最低基本介质缓冲与 HEPES 到每一个井。让板在室温下稳定 10 分钟。同时，通过将100X基板的一体积与19卷LSS稀释缓冲液相结合，准备毛茸茸基材，形成5倍的基材，与细胞培养基混合。当细胞准备就绪时，将25微升的库存添加到每个井中，轻轻混合板10秒钟。然后在室温下测量发光10分钟，以稳定信号。对于配体加法的实验，在经过修改的 Eagle 使用 HEPES 缓冲的最小基本介质中准备 13.5 倍配体解决方案，并使用多通道移液器每井添加 10 微升。然后用手或轨道摇床混合板。该协议已成功用于研究原型 GPCR 和两个 β-逮捕素等同项形式之间的相互作用。筛选出四种不同的质粒组合，并选择了发光信号最高的质粒组合进行进一步实验。每种β-阻滞素等等物的EC50值都是使用剂量-反应曲线确定的，这些曲线是从动力学研究中每个浓度的最大反应中获得的。在设计 PCR 底件时，要特别注意这一点至关重要。正确的结构的发展对于这些测定的成功至关重要。按照这种方法，您将能够监测受体-β-卡丁蛋白的相互作用，以及受体与其他细胞蛋白的相互作用。这种方法将有助于回答GPCR药理学领域的基本问题，通过研究GPCR-β-阻滞剂相互作用如何通过改变配体结构来调节。

Research

JoVE Journal

Biochemistry

Monitoring GPCR-&#946;-arrestin1/2 Interactions in Real Time Living Systems to Accelerate Drug Discovery

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

Preparation and In Vivo Use of an Activity-based Probe for N-acylethanolamine Acid Amidase

制备及<em&gt;在体内</em&gt;使用基于活动的探针对<em&gt;ñ</em&gt; -acylethanolamine酸酰胺酶

Activity-based protein profiling (ABPP) is a method for the identification of an enzyme of interest in a complex proteome through the use of a chemical ...

科研

生物化学

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

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. ...

Real-time Tracking of DNA Fragment Separation by Smartphone

通过智能手机实时跟踪DNA片段分离

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Regeneration of Arrayed Gold Microelectrodes Equipped for a Real-Time Cell Analyzer

用于实时细胞分析仪的阵列金电极再生

The label-free cell-based assay is advantageous for biochemical study because of it does not require the use of experimental animals. Due to its ability ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

低亲和力受体-配体相互作用的细胞外蛋白微阵列技术

Secreted factors, membrane-tethered receptors, and their interacting partners are main regulators of cellular communication and initiation of signaling ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

放射性脉冲追逐对活细胞蛋白质折叠、迁移和降解的分析

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

rad51 介导的 dna 链交换反应的实时观测

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

用于膜蛋白-蛋白质相互作用分析的原生细胞膜纳米粒子系统

Protein-protein interactions in cell membrane systems play crucial roles in a wide range of biological processes- from cell-to-cell interactions to signal ...

Measuring Nucleotide Binding to Intact, Functional Membrane Proteins in Real Time

实时测量核苷酸与完整功能性膜蛋白的结合

We have developed a method to measure binding of adenine nucleotides to intact, functional transmembrane receptors in a cellular or membrane environment. ...

Enabling Real-Time Compensation in Fast Photochemical Oxidations of Proteins for the Determination of Protein Topography Changes

实现蛋白质快速光化学氧化的实时补偿，用于确定蛋白质地形变化

Fast photochemical oxidation of proteins (FPOP) is a mass spectrometry-based structural biology technique that probes the solvent-accessible surface area ...