Name: fluorescence anisotropy as a tool to study protein-protein interactions
Uploaded: 2016-10-21T15:00:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

Authors acknowledge the financial support from CONACyT project numbers 167359 and 177138, and from DGAPA-UNAM project number IN201615.

1. SBDS闪光标签蛋白表达纯化注:对于各向异性的实验中，对应于半胱氨酸 - 半胱氨酸-Pro的基-Gly-半胱氨酸 - 半胱氨酸加入到人类SBDS通过PCR编码序列的C-末端序列的闪光标签。此构建物亚克隆到表达载体pRSET-A，并转化到大肠杆菌 C41细胞以表达编码N-末端六组氨酸标签（His标签）的蛋白质，人类SBDS编码序列和C-末端闪光标签10。 <ol><li> SBDS-FLASH蛋白的表达 <ol><li>转化感受态大肠杆菌 C41细胞，使用标准的热休克协议19质粒PRSET-HisSBDS闪光。板中补充有100微克/毫升氨苄青霉素固体的Luria-BERTANI（LB）培养基中的细胞。的LB固体培养基组合物包括10个克NaCl，5g酵母提取物，10g胰蛋白胨和1升20体积克琼脂的。 </li><li>在37℃下培养转化的细菌，直到吸光度在600nm（A 600）达到在1升补充有100微克/毫升氨苄青霉素的LB液体培养基的0.5-0.7。 </li><li>通过加入0.5mM的异丙基β-D-1-硫代半乳糖苷至培养诱导蛋白质表达，并继续进行另外5小时孵育。 </li><li>收集经离心细菌悬浮液3800×g离心在4℃下10分钟。去除上清。在这一点上，无论是存储的细胞沉淀在-20℃或立即用于蛋白质纯化。 </li></ol></li><li> SBDS-FLASH蛋白纯化 注意:使用快速蛋白质液相色谱（FPLC）系统或蠕动泵执行的所有层析步骤。镍离子色谱-affinity用5ml的快速流动柱。阴离子交换层析用5ml的磺强阳离子交换柱。 3毫升/分钟的流速，使用分的所有的色谱步骤。 <ol><li>悬浮细胞在35毫升SBDS裂解的Buffer通过超声处理用10秒ON和30秒的循环4分钟关的总时间补充有1mM苯甲基磺酰氟（PMSF）和裂解（50mM磷酸盐缓冲液pH 7.5，300mM NaCl的20 mM咪唑）中，在4 C。 </li><li>离心样品以9000rpm×g离心在4℃下50分钟。 </li><li>保持上清液弃沉淀以除去细胞碎片。 </li><li>平衡用SBDS裂解缓冲液3倍柱体积（CV）的镍离子亲和层析柱和引入澄清的上清液至色谱柱上的整体。 </li><li>通过洗涤3的CV SBDS裂解缓冲液的除去未结合的蛋白质，并用3 CV SBDS洗脱缓冲液（50mM磷酸盐缓冲液pH 7.5，300mM NaCl的，​​250mM咪唑）洗脱。 </li><li>稀释洗脱的蛋白6倍，用50mM磷酸盐缓冲液pH 6.5，并通过一个0.22微米的纤维素膜除去通过过滤可能聚集体。 </li><li>用3 CV低盐S列中的平衡的磺基阳离子交换柱缓冲液（50mM磷酸盐缓冲液pH 6.5，50mM NaCl中），并引入来自前面步骤的蛋白质样品。 </li><li>洗涤未结合的物质用3的CV低盐S柱缓冲液和洗脱蛋白在一个步骤中，用50mM磷酸盐缓冲液pH 6.5，1M NaCl洗涤。 </li><li>稀释洗脱的蛋白3.3倍，用50mM磷酸盐缓冲液pH 6.5。离心3800 xg离心浓缩用超滤装置蛋白质15分钟。闪光灯冻结蛋白在液氮中，并储存在-80℃直至进一步使用。 </li><li>验证的蛋白质通过SDS-PAGE分析和考马斯染色20的纯度。 </li></ol></li></ol> 2. EFL1蛋白表达纯化注:EFL1是在加1/10发散子21和酿酒酵母垫上 3&#39;UTR在载体pRS426的调节下表达。重组蛋白编码融合到烟草蚀纹病毒蛋白酶人类EFL1同种型1（TEV）识别位点，并在C末端六组氨酸标签。 <ol><li> EFL1蛋白的表达 <ol><li>变换S.酵母 BCY123细胞使用标准乙酸锂协议22质粒pRS426-EFL1TevHis。板的所有合成滴出媒体的转化的细胞，而不补充有2％（重量/体积）葡萄糖尿嘧啶（SD-URA）。对SD-URA培养基组合物包括8克酵母氮碱无氨基酸下，将11g酪蛋白氨基酸，55毫克硫酸腺嘌呤，55毫克酪氨酸，60毫克亮氨酸和60mg色氨酸为1升体积。 </li><li>培养在30℃下转化的酵母，直至A 600达到1.8在1升的SD-URA培养基补充有0.5％（重量/体积）的葡萄糖。 </li><li>通过加入2.8％（重量/体积）的半乳糖的培养诱导蛋白质表达，并继续进行另外18小时的温育在30℃。 </li><li>收集通过离心分离酵母悬浮液3800×g离心在4℃下10分钟。去除上清。在这一点上，无论是存储的细胞沉淀在-20℃或立即用于蛋白质纯化。 </li></ol></li><li> EFL1蛋白纯化 注意:使用FPLC系统或蠕动泵被执行的所有层析步骤。镍离子 -affinity色谱以3毫升/分钟的流速使用5ml的快速流动柱。尺寸排阻色谱法使用的125ml列预填充的Superdex 200树脂以1毫升/分钟的流速。 <ol><li>悬浮细胞在50ml补充有1mM的PMSF和1mM苯甲脒EFL1裂解缓冲液（50mM的Tris-HCl pH值为8，300mM NaCl的20 mM咪唑，5mM的MgCl 2的，10％甘油）中，并通过破坏细胞摩擦使用玻璃珠（直径= 0.5mm）的为2分钟开，15分钟关6分钟使用周期的总时间，在4℃的珠打浆机。 </li><li>离心样品以9000rpm×g离心在4℃下50分钟。 </li><li>保持上清液弃沉淀以除去细胞碎片。 </LI&gt; <li> 3 CV EFL1裂解液平衡的镍离子亲和柱，并介绍所有的澄清上清上柱。 </li><li>通过洗涤3的CV EFL1裂解缓冲液的除去未结合的蛋白质，并用3 CV EFL1洗脱缓冲液（50mM的Tris-盐酸pH值为8，300mM NaCl的，250mM咪唑，5mM的MgCl 2的，10％甘油）洗脱。 </li><li>用各向异性缓冲的1.5 CV（50毫摩尔Tris-盐酸pH值7.5，300mM NaCl的，5mM的MgCl 2的，10％的甘油，5mM的β巯基乙醇）平衡尺寸排阻柱。 </li><li>浓缩到1ml 3800 xg离心从由离心超滤装置在Ni 2+亲和层析柱洗脱，所需体积的EFL1蛋白。引进的大小排阻柱样品。 </li><li>收集被洗脱的蛋白质并通过超滤浓缩至约30微米的最终浓度。闪冻在液氮中，并存储该蛋白质在-80℃直至进一步使用。验证次通过SDS-PAGE分析和考马斯染色20蛋白质电子纯度。 </li></ol></li></ol> 3. SBDS-FLASH的标签与Flash荧光染料4&#39;，5&#39;-双（1,3,2 dithioarsolan -2-基）荧光素<ol><li>混合SBDS-FLASH蛋白3纳摩尔与4&#39;的3纳摩尔，5&#39;-双（1,3,2- dithioarsolan -2-基）在各向异性缓冲液5微升体积的荧光素染料。 </li><li>让反应继续进行，在4℃下8小时。透析针对各向异性缓冲器样品过夜，以除去游离的染料。 </li><li>使用朗伯 - 比尔定律量化标记蛋白的％。测量在280nm和在用适当体积的石英比色皿分光光度计508处的吸光度。注:考虑以下摩尔吸光系数（M -1厘米-1）: <img alt="Equation1B" src="/files/ftp_upload/54640/54640eq1B.jpg" /></li><li>计算使用公式2中标记SBDS-FLASH蛋白的浓度。 <img alt="方程2" src="/files/ftp_upload/54640/54640eq2.jpg" /></li><li>通过从先前的步骤代替计算值:C SBDS闪光计算使用公式3总SBDS蛋白的浓度。 <img alt="Equation3" src="/files/ftp_upload/54640/54640eq3.jpg" /></li><li>计算使用公式4标记的蛋白质的百分比。 <img alt="公式4" src="/files/ftp_upload/54640/54640eq4.jpg" /></li></ol> 4.荧光各向异性实验注:各向异性实验在装备有使用在设备中的软件提供的各向异性程序执行了极化工具箱和数据收集分光光度计进行。激发波长为494纳米的为8纳米的光谱带宽，并使用530±25nm的带通滤波器的发射被记录下来。测量在25℃下在200微升比色杯中进行用一个5毫米的路径长度10。 <ol><li><html>在荧光比色皿，将200微升30纳米SBDS闪各向异性缓冲液和滴定2微升30微米EFL1的。调匀，让站在反应3分钟测量各向异性值之前。 </li><li>重复步骤4.1至40微升EFL1的总体积已被添加。 </li></ol> 5.数据分析<ol><li>利用非线性最小二乘回归算法拟合数据到合适的装订模式。对于最常见的结合模型方程表示在表1中。 </li><li>评价，通过检查拟合23的残差描述了蛋白质之间的相互作用的最佳模式。支持额外的实验选择的模型。 </li></ol> 表1普通的蛋白质-蛋白质相互作用结合模型和描述它们的数学方程。0 / 54640table1large.jpg"目标="_空白"&gt;请点击这里查看此表的放大版本。 <img alt="表格1" src="/files/ftp_upload/54640/54640table1.jpg" />

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M., Jezewska, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+Intensity,+Anisotropy,+and+Transient+Dynamic+Quenching+Stopped-Flow+Kinetics.">Fluorescence Intensity, Anisotropy, and Transient Dynamic Quenching Stopped-Flow Kinetics.</a> Spectroscopic Methods of Analysis. 875, 105-133 (2012).</li><li>Asano, N., 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=Direct+interaction+between+EFL1+and+SBDS+is+mediated+by+an+intrinsically+disordered+insertion+domain.">Direct interaction between EFL1 and SBDS is mediated by an intrinsically disordered insertion domain.</a> Biochem Biophys Res Commun. 443 (4), 1251-1256 (2014).</li></ol>

The authors have nothing to disclose and they have no competing financial interests.

与蛋白质大多数生化实验不仅需要纯的蛋白质，而且大量的它们，而不管使用的技术。由于这个原因，用于这种类型的实验的蛋白质被异源表达得到，因为它是这里提出的情况。花期光谱需要荧光团的研究分子中的存在。芳香族残基构成的蛋白质的固有荧光团，但是，使用其信号来研究蛋白质 - 蛋白质相互作用变得复杂分析，因为它们是共同的大多数蛋白质和将存在于两个，受体蛋白和配体。此?...

细胞含有生物大分子是不断地与彼此交互的一许多。该协会产生了参与负责其在信号转导，基因表达和除其他细胞迁移的调节功能的细胞通路的复合物。发生在一个小区中的所有蛋白质 - 蛋白质相互作用包括称为相互作用组的网络。在酿酒酵母中的蛋白质的70％以上已被证明有相互作用伙伴1。理解的细胞的相互作用组和它们的功能提供关于复杂性和生物多样性的相关信息。数种方法已经描述于鉴定和表征蛋白质 - 蛋白质相互作用。不同的高通过放的方法，如酵母双杂交2，蛋白质片段互补测定法如图3所示 ，耦合到质谱和蛋白microarra亲和纯化4YS用于识别交互5,6。一旦确定，就必须对其进行验证，这可能在逐案的基础而改变。通常，这些实验涉及破坏相互作用本身在该相互作用对， 例如的个别成员的水平，通过基因缺失或蛋白之一的表达，然后寻找在属性或改变的其它部件的功能细胞水平。随后，生物物理技术7用于在分子水平来表征蛋白质-蛋白质相互作用。为此，虽然量热法和荧光光谱被用于定量和机械地描述它们的蛋白质复合物的结构通过X射线晶体学，核磁共振和低温电子显微镜来确定。 在这项工作中，荧光各向异性用作技术来表征GTP酶EFL1和SBD之间的相互作用S蛋白。这些蛋白质通过来自60S核糖体亚基8的表面促进真核起始因子6的释放参与核糖体的合成。该SBDS蛋白在称为Shwachman-Diamond综合征9和充当EFL1减小为鸟苷二磷酸10,11其亲和性的鸟嘌呤核苷酸交换因子的疾病的突变。在SBDS疾病的突变废除与EFL1的相互作用，从而防止其激活。 荧光各向异性在生物应用通常用于研究蛋白质 - 肽或蛋白质 - 核酸相互作用。它是基于荧光团与偏振光产生一个部分偏振的发射激发的原理。荧光各向异性由等式1定义: <img alt="公式1" src="/files/ftp_upload/54640/54640eq1.jpg" />在那里我VV和VH我是垂直（VV）和水平（V H）的荧光强度偏振的发射时，样品被激发与垂直偏振光12。荧光各向异性是影响荧光团的旋转扩散速度的因素敏感，因而取决于温度，溶液的粘度和荧光团的表观分子大小。含有荧光团的增加，当它与另一蛋白和这种变化相互作用的蛋白质的表观大小然后可以作为各向异性的变化进行评估。更具体地说，在相对于它的荧光寿命溶液缓慢旋转的荧光团将有一个大I VV值和小我的VH的值，因此，将表现出相对大的各向异性。对于迅速相对于它们的荧光寿命滚入荧光团，我VV和VH我将类似和各向异性值将是12小</sup&gt;（ 图1）。此外，有一个良好的各向异性信号到噪声测量，有必要有一个与相似于感兴趣的分子的旋转相关时间荧光寿命的荧光团。否则，就不可能准确地记录游离蛋白在复之间以及在各向异性的差异。例如，荧光探针与接近4纳秒如荧光素或若丹明附着到100道尔顿的低分子量化合物一生各向异性为0.05。绑定到160 kDa的将其各向异性值提高到0.29的分子;可精确地测量的差。与此相反，参与结合反应，其增加的分子大小而变化，从65至1000 kDa的相同荧光探针只会造成0.28各向异性变化到0.3，这是太小而不能精确测量。在这种情况下，用400纳秒的使用寿命的探针会更适合12。 <p类="jove_content"&gt; <img alt="图1" src="/files/ftp_upload/54640/54640fig1.jpg" /> 图1.用于测量荧光各向异性和过程的设备的示意图。用于执行蛋白质-蛋白质相互作用的实验测量荧光各向异性的设备的示意图。滚入快速显示小各向异性结合后相互作用的合作伙伴增加了荧光。 <a href="http://ecsource.jove.com/files/ftp_upload/54640/54640fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 荧光应用需要荧光团在任何所研究的分子的存在。研究蛋白质 - 蛋白质相互作用有三种类型的荧光团:1）中存在的蛋白质中的色氨酸残基，2）化学连接的荧光团和3）荧光融合伙伴，例如绿色荧光蛋白（GFP）及其deriva表3-6。大多数蛋白质具有在其结构色氨酸残基，从而来测量相互作用的最简单的方法是通过监视在相应的荧光光谱的变化或通过在色氨酸残基的荧光强度监视更改。然而，色氨酸残基可存在于两种蛋白质的分析复杂化。另一方面，对于荧光团以改变其荧光性质由于交互它需要被位于或结合位点附近，它可以与交互本身干涉。使用笨重的荧光，如GFP时，这种需要特别注意。如果没有这些荧光团可用于结合研究是必要的，那么，引入外源性荧光团所涉及的蛋白之一。许多化学合成的荧光团存在，并且可被共价连接到通过其反应性基团的蛋白质，如胺基（赖氨酸或N末端的侧链）和在半胱氨酸的硫醇基团。 F与异硫氰酸酯和琥珀酰亚胺酯luorophore衍生物与酰胺基团反应而碘乙酰胺和马来酰亚胺是硫醇反应性基团13。在荧光应用中最常用的染料是荧光素的衍生物，和若丹明绿染料，香豆素，BODIPY荧光团和Alexa Fluor染料。市售的荧光团及其使用的详细列表可在参考文献14,15找到。对于成功的标记，反应性基团必须在蛋白质的表面上暴露出来，但由于大量通常存在于多肽的反应性官能团的是很难得到的位点特异性修饰。在这项研究的目的蛋白质，SBDS，含有5游离半胱氨酸和33的赖氨酸，可能导致在多个位点标记。非均匀标签可能影响结合和不同的荧光分子可能结合后引起不同的荧光强度信号将复杂的数据分析。奥雅纳rcome这一问题，我们使用了闪光灯荧光团，4&#39;，5&#39;-双（1,3,2- dithioarsolan -2-基）荧光素到网站直接标记的SBDS蛋白。这是一arsenoxide染料与在一个基序知道作为由序列CCXXCC，其中X是除半胱氨酸16,17之外的任何氨基酸的闪光标记为四个间隔半胱氨酸的高亲和性。这四半胱氨酸基序被添加到通过遗传工程N-或蛋白质的C末端与适当的接头，以防止蛋白质的总体折叠的破坏在一起。由闪光染料和Flash标签的一对最初设计为位点特异性标签的蛋白在活细胞中17，但它也可以使用，因为它是这里例举标记纯化的蛋白在体外 。此外，酶的策略也已经被开发，以使蛋白18的位点特异性官能化。 在这个手稿中，我们描述的荧光各向异性的用处山工具来研究蛋白质 - 蛋白质相互作用。结合可以通过结合曲线形状的简单检查来评估而定量信息可以从实验数据的拟合而获得。

<table><tbody><tr><td>0.5 mm Glass beads</td><td>Biospec Products</td><td>11079105</td><td></td></tr><tr><td>Tris Base</td><td>Formedium</td><td>TRIS01</td><td>Ultra pure</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>dye 4&amp;rsquo;,5&amp;rsquo;-bis(1,3,2 dithioarsolan-2-yl) fluorescein</td><td>ThermoFischer Scientific</td><td>LC6090</td><td>This kit contains the dye to label a FlAsH tag</td></tr><tr><td>Ampiciline</td><td>IBI Shelton Scientific, Inc</td><td>IB02040</td><td></td></tr><tr><td>D(+)-Glucose Anhydrous</td><td>Formedium</td><td>GLU03</td><td></td></tr><tr><td>D(+)-Galactose</td><td>Formedium</td><td>GAL03</td><td></td></tr><tr><td>L-Leucine</td><td>Formedium</td><td>DOC0157</td><td></td></tr><tr><td>L-Tryptofan&amp;nbsp;</td><td>Formedium</td><td>DOC0189</td><td></td></tr><tr><td>Bezamidine hydrochloride</td><td>Sigma-Aldrich</td><td>B6506-5G</td><td></td></tr><tr><td>PMSF</td><td>Gold Biotechnology, Inc</td><td>P-470-25</td><td>Phenylmethylsulfonyl fluoride</td></tr><tr><td>NaCl</td><td>Formedium</td><td>NAC02</td><td>Sodium Chloride&amp;nbsp;</td></tr><tr><td>Glycerol</td><td>Tecsiquim, S.A. de C.V.</td><td>GT1980-6</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck Millipore Corporation</td><td>1725711000</td><td>Magnesium Chloride</td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399-500G</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Sigma-Aldrich</td><td>M6250-100ML</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P3786-500G</td><td>Potassium phosphate dibasic</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S3139-500G</td><td>Sodium phosphate monobasic</td></tr><tr><td>Yeast nitrogen base without amino acids</td><td>Formedium</td><td>CYN0410</td><td></td></tr><tr><td>Yeast extract</td><td>Formedium</td><td>YEM03</td><td>Micro Granulated</td></tr><tr><td>L-Tyroisne</td><td>Formedium</td><td>DOC0193</td><td></td></tr><tr><td>Adenine sulphate</td><td>Formedium</td><td>DOC0230</td><td></td></tr><tr><td>Casamino acids</td><td>Formedium</td><td>CAS03</td><td></td></tr><tr><td>Tryptone</td><td>IBI Shelton Scientific, Inc</td><td>IB49182</td><td></td></tr><tr><td>IPTG</td><td>Formedium</td><td>IPTG025</td><td></td></tr><tr><td>Filtration units</td><td>Merck Millipore Corporation</td><td>UFC901096</td><td>Amicon Ultra-15, membrana PLGC Ultracel-PL, 10&amp;nbsp;kDa</td></tr><tr><td>Membrane Filter</td><td>Merck Millipore Corporation</td><td>GSWP04700</td><td>Membrane Filter, mixed cellulose esters, Hydrophilic, 0.22&amp;nbsp;&amp;micro;m, 47&amp;nbsp;mm, white, plain</td></tr><tr><td>Ni&lt;sup&gt;2+&lt;/sup&gt; affinity column</td><td>QIAGEN</td><td>30760</td><td>Cartridge pre-filled with 5&amp;nbsp;ml Ni-NTA Superflow</td></tr><tr><td>Strong Sulfopropyl cation exchanger column</td><td>GE Healthcare Life Science</td><td>17-5157-01</td><td>HiTrap SP Sepharose FF 5 ml</td></tr><tr><td>Size Exclusion column</td><td>GE Healthcare Life Science</td><td>28989335</td><td>HiLoad 16/600 Superdex 200 PG</td></tr><tr><td>Fluorescence cell</td><td>Hellma Analytics</td><td>111-057-40</td><td></td></tr><tr><td>Spectrophotometer</td><td>Agilent Technologies</td><td>G6860AA</td><td>Cary 60 UV-Vis</td></tr><tr><td>Shaker</td><td>ThermoFischer Scientific</td><td>SHKA4000-7</td><td>MaxQ 4000 Benchtop temperature range Ambient-15&amp;deg; to 60&amp;deg;C</td></tr><tr><td>Centrifuge</td><td>ThermoFischer Scientific</td><td>75004271</td><td>Heraeus Megafuge 16R</td></tr><tr><td>FPLC</td><td>Pharmacia Biotech</td><td>Discontinued</td><td>FPLC system conductivity UV-MM II monitor P500 pump fraction</td></tr><tr><td>Spectrofluorometer</td><td>Olis</td><td>No applicable</td><td>Olis DM 45 with Polarization Toolbox</td></tr></tbody></table>

fluorescence anisotropy as a tool to study protein-protein interactions

蛋白质 - 蛋白质相互作用发挥活生物体的功能中起重要作用。一旦相互作用已被确定和验证是必要在结构和机械水平来表征它。几个生物化学和生物物理方法用于此目的存在。其中，荧光各向异性，特别使用一种强大的技术，当荧光团标记蛋白质的荧光强度保持在蛋白质 - 蛋白质相互作用常数。在该技术中，荧光团标记的蛋白是激发选择性地激发根据其与入射光束的相对取向的荧光团的子集的适当波长的垂直偏振光。所得发射还具有方向性，其在垂直和水平平面关系定义各向异性（r）的如下:R =（I VV -I V H）/（I VV + 2I V H），其中I VV和我<sUB&gt; VH是垂直和水平分量的荧光强度分别。荧光各向异性是荧光团，即连接于蛋白，它是建立在蛋白质 - 蛋白质相互作用改变荧光团的表观分子大小的旋转扩散敏感。在本文中，使用荧光各向异性作为一种工具来研究蛋白质相互作用是例证，以解决Shwachman-Diamond综合征（SBDS）突变蛋白质和延伸因子样-1 GTP酶（EFL1）之间的绑定。传统上，用荧光团的蛋白质的标记上的硫醇基（半胱氨酸）或在蛋白质的氨基（N-末端胺或赖氨酸）进行。然而，SBDS拥有多项半胱氨酸和赖氨酸并没有让现场指挥它的标签。作为一种替代技术，染料4&#39;，5&#39;-双（1,3,2- dithioarsolan -2-基）荧光素用于特异性标记一个四半胱氨酸基序，Cys-的Cys-Pro的基-Gly-半胱氨酸 - 半胱氨酸，在重组SBDS蛋白的C末端遗传工程。拟合实验数据提供了关于这些蛋白质之间的结合模式的数量和机械信息。

执行任何各向异性实验它，因为物质的混合物所观察到的各向异性由等式5表示的，以排除在荧光团的荧光强度大的变化是很重要的: <img alt="Equation5" src="/files/ftp_upload/54640/54640eq5.jpg" />其中F i表示各成分及r的分数荧光i是对应的各向异性。各组分?...

Watch this Scientific Journal Video about Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions at JoVE.com

荧光各向异性作为研究蛋白质-蛋白质相互作用的工具|生物化学|视频

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

蛋白质相互作用是在一个细胞的功能的心脏。量热和光谱技术通常用于表征它们。这里我们介绍荧光各向异性作为一种工具来研究在Shwachman-Diamond综合征（SBDS）突变蛋白质和延伸因子样1 GTP酶（EFL1）之间的相互作用。

荧光各向异性作为一种工具来研究蛋白质相互作用

Protein-protein interactions play an essential role in the function of a living organism. Once an interaction has been identified and validated it is ...

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30.4K 次观看.  Universidad Nacional Autónoma de México. 蛋白质相互作用是在一个细胞的功能的心脏。量热和光谱技术通常用于表征它们。这里我们介绍荧光各向异性作为一种工具来研究在Shwachman-Diamond综合征（SBDS）突变蛋白质和延伸因子样1 GTP酶（EFL1）之间的相互作用。 

视频: Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Utilizing the Ethylene-releasing Compound, 2-Chloroethylphosphonic Acid, as a Tool to Study Ethylene Response in Bacteria

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ripening. Exogenous ethylene applied during early plant development triggers the triple response phenotype; a shorter and thicker hypocotyl with an exaggerated apical hook. Despite the intimate relationship between plants and bacteria, the effect of exogenous ethylene on bacteria has been greatly overlooked. This is partly due to the difficulty of controlling gaseous ethylene within the laboratory without specialized equipment. 2-Chloroethylphosphonic acid (CEPA) is a compound that decomposes into ethylene, chlorine, and phosphate in a 1:1:1:1 molar ratio when dissolved in an aqueous medium of pH 3.5 or greater. Here we describe the use of CEPA to produce in situ ethylene for the investigation of ethylene response in bacteria using the fruit-associated, cellulose-producing bacterium Komagataeibacter xylinus as a model organism. The protocols described herein include both the verification of ethylene production from CEPA via the Arabidopsis thaliana triple response assay and the effects of exogenous ethylene on K. xylinus cellulose production, pellicle properties and colonial morphology. These protocols can be adapted to examine the effect of ethylene on other microbes using appropriate growth media and phenotype analyses. The use of CEPA provides researchers with a simple and efficient alternative to pure ethylene gas for the routine determination of bacterial ethylene response.

The protocols outlined herein facilitate the convenient investigation of bacterial ethylene responses by utilizing 2-chloroethylphosphonic acid (CEPA). Ethylene is produced in situ through the decomposition of CEPA in an aqueous bacterial growth medium, circumventing the requirement for pure ethylene gas.

利用乙烯释放化合物，2-氯乙基膦酸，作为一种工具来研究细菌乙烯反应

Ethylene (C2H4) is a gaseous phytohormone that is involved in numerous aspects of plant development, playing a dominant role in senescence and fruit ...

科研

生物化学

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em&gt;体外</em

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

应用三色单分子焦虑研究蛋白质相互作用的相关性

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be employed using a conventional confocal microscope equipped with digital detectors. A protocol for the use of the technique in vitro is shown by means of a use case where number and brightness can be seen to accurately quantify the oligomeric state of mVenus-labelled FKBP12F36V before and after the addition of the dimerizing drug AP20187. The importance of using the correct microscope acquisition parameters and the correct data preprocessing and analysis methods are discussed. In particular, the importance of the choice of photobleaching correction is stressed. This inexpensive method can be employed to study protein-protein interactions in many biological contexts.

Calibration-free In Vitro Quantification of Protein Homo-oligomerization Using Commercial Instrumentation and Free, Open Source Brightness Analysis Software

This protocol describes a calibration-free approach for quantifying protein homo-oligomerization in vitro based on fluorescence fluctuation spectroscopy using commercial light scanning microscopy. The correct acquisition settings and analysis methods are shown.

利用商用仪器和自由开源亮度分析软件对蛋白质的聚齐聚进行无标定的体外定量化

Number and brightness is a calibration-free fluorescence fluctuation spectroscopy (FFS) technique for detecting protein homo-oligomerization. It can be ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the local flexibility and dynamics of the deoxyribonucleic acid (DNA)-binding forkhead (FKH) domain of the FoxP1 transcription factor. FoxP1 dimerizes through a three-dimensional domain-swapping (3D-DS) mechanism, forming a disordered intermediate with or without DNA. Since 3D-DS involves an intrinsically disordered region, understanding its behavior is crucial for elucidating the structural and functional properties of FoxP1. Using a single-cysteine-labeled FoxP1, we conducted single-molecule fluorescence anisotropy (smFA) experiments, applying dynamic anisotropy Photon Distribution Analysis (daPDA) and time-resolved anisotropy Burst Variance Analysis (traBVA) approaches to probe local flexibility and dynamics. This protocol provides a detailed, step-by-step guide for smFA measurements, emphasizing time-resolved analyses, variance, and probability distribution techniques to capture structural dynamics across different timescales. This approach enabled us to relate dynamics and heterogeneity to FoxP1 dimerization and DNA binding, highlighting the complex action mechanism that characterizes this transcription factor.

Here, we present the protocol to study the local flexibility and dynamics of biomolecules using time-resolved fluorescence anisotropy at the single-molecule level in confocal microscopy mode.

We describe a protocol for conducting time-resolved fluorescence anisotropy at the single-molecule level using confocal microscopy to investigate the ...

Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

Source: Jung, C. et al., <a href="https://app.jove.com/v/58763">High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy.</a> J. Vis. Exp. (2019).
This video describes high-performance fluorescence anisotropy that helps to monitor the interaction between transcription factors and DNA. The assay monitors the interactions of a fluorophore-labeled DNA molecule with its specific transcription factor by measuring the degree of polarization due to molecular rotation or anisotropy of the fluorophore-labeled DNA.

用于确定转录因子-DNA 结合亲和力的荧光各向异性

Source: Jung, C. et al., High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence ...

JoVE Encyclopedia of Experiments

Biological Techniques

Biomolecular Interaction Detection Techniques

Protein-nucleic acid interactions

Fluorescence Anisotropy-Based Detection of Protein-Protein Interactions

Mix 3 nanomoles of the SBDS-FlAsH protein with 3 nanomoles of the Lumio Green dye in a 5-microliter volume of anisotropy buffer. Let the reaction proceed for 8 hours at 4 degrees Celsius. After 8 hours, dialyze the sample against the anisotropy buffer overnight to remove the free dye. Measure the absorbance at 280 nanometers and 508 nanometers in a spectrophotometer using a quartz cuvette. Then, use the Lambert-Beer law to quantify the percent of labeled protein as described in the text protocol.
Before measuring the anisotropy value, each titration step of the protein-ligand should be done carefully, ensuring that the entire sample is dispensed into the solution, and becomes homogeneous.
In a fluorescence cuvette, place 200 microliters of 30 nanomolar SBDS-FlAsH in anisotropy buffer, and titrate 2 microliters of 30 micromolar EFL1. Mix thoroughly, and let the reaction stand for 3 minutes before measuring the anisotropy and fluorescence value. Repeat this process until a total volume of 40 microliters of EFL1 has been added. As a final step, fit the data to a presumed binding model, as described in the text protocol.

基于荧光各向异性的蛋白质-蛋白质相互作用检测

Protein-protein interactions

EoE Sub Articles

Fluorescence Fluctuation Spectroscopy to Study Protein Homo-Oligomerization

To set up the multiwell plate array, first, prepare a solution of 100 nanomolar purified FKBP12 in the same buffer used for size exclusion chromatography. Sonicate and centrifuge with a quick spin of 13,000 RPM to prevent the formation of aggregates.
Now, pipette 100 to 200 microliters of the diluted protein into an 8-well observation chamber with a glass bottom. Add the BB dimerizer to final concentrations of 10, 20, 40, 80, 100, 150, 300, and 500 nanomolar. As a reference, prepare solution of 100 nanomolar mVenus alone to evaluate potential aggregation and precipitation effects, and to recover a brightness value for the monomer with the same acquisition settings.
Any light scanning microscope confocal system equipped with digital detectors or well-characterized analog detectors, and capable of keeping a constant dwell time for every pixel acquired can be used. Select the collar correction water immersion objective designed for fluorescence correlation spectroscopy.
Now add a drop of water to the collar correction water immersion objective. Mount the 8-well observation chamber into the stage. Set the excitation beam path by turning on the 514-nanometer laser, and setting it at 20 to 100 nanowatts power at the exit of the objective. Turn on one HyD detector. Detectors capable of photon-counting are preferable. Select the emission window from 520 to 560 nanometers.
For the acquisition mode, use 16 by 16 pixels.
Set the pixel dwell time such that the frame time is longer than the protein diffusion, and the pixel dwell time is much shorter. This corresponded to setting the dwell time to approximately 13 microseconds for the system used in this demonstration.
Set the pinhole at one Airy unit for the corresponding emission of approximately 545 nanometers. Select the xy-time acquisition mode and select the number of frames to be acquired per acquisition and well. Now, set the pixel size at approximately 120 nanometers.
If the system is equipped with high-throughput mode, introduce the coordinates of each well, and the number of acquisitions per well to automate the process. If the system is equipped with a perfusion system, load the BB solution, and program the perfusion to start right after the 5,000th frame to evaluate the kinetics of dimerization while acquiring 10,000 images.
Select the correct well, and focus on the solution. Then, start the acquisition and save the resulting stack of images in TIFF format.

荧光波动光谱研究蛋白质同源寡聚化

Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts

Transfer the cell solution of one well to the corresponding well. Mix gently by pipetting a few times up and down, then, seed the mixed cells on a 35-milliliter glass-bottomed dish, and culture the seeded cells at 37 degrees Celsius and 5% carbon dioxide for one day.
In the laser-scanning confocal microscope software, set up the optical path. To avoid spectral cross-talk, select two separate tracks to excite and detect mEGFP or mEYFP and mCherry or mCardinal sequentially, and select &#34;Switch Tracks Every Line.&#34; For the detection, use appropriate filters for both channels.
Place the dish containing the mixed cells on the sample holder. After waiting 10 minutes to ensure temperature equilibration and to reduce focus drift, focus on the cells using the Transmission Light in the &#34;Locate&#34; menu. Search for a pair of &#39;red&#39; and &#39;green&#39; cells in contact with each other.
Next, select a scan path perpendicular to cell-cell contact using the &#34;Crop&#34; button. &#34;Zoom&#34; to achieve a pixel size of 50 to 200 nanometers and select &#34;Line&#34; in &#34;Scan Mode.&#34; Set &#34;Frame Size&#34; to 128 by 1 pixels. Set &#34;Scan Speed&#34; to the maximum allowed value, then set cycles to 100,000 to 500,000.
Following this, choose the appropriate laser powers. Set the detectors to &#34;Photon Counting&#34; Mode. Press &#34;Start Experiment&#34; to start the acquisition.

荧光各向异性作为一种工具来研究蛋白质相互作用

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