Name: luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells
Uploaded: 2014-09-16T14:00:00.000+00:00
Duration: 8 min 31 s
Description: Watch this Scientific Journal Video about Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells at JoVE.com

这项工作是由卫生部授予GM094246，美国心脏协会授予11GRNT7890004，以及美国国家科学基金会资助的MCB-1110501国家机构的支持。

1，创建构建含利息的蛋白质克隆表达目的蛋白到合适的载体的基因。使用载体如pcDNA系列或pRK5，因为它们非常适合用于在哺乳动物系统，如HEK293和CHO细胞中表达。 2，选择网站上的蛋白与荧光基团进行标记<ol><li>选择标签的网站，都能够体现出蛋白质中可能的构象变化。如果可能的话，使用同源蛋白的蛋白质或晶体结构，以帮助做出此决定。如果没有晶体结构信息，可以使用在线软件来预测蛋白质的可能的结构，从而获得洞察适当部位。 </li><li>选择的残基，使得所选择的残基的侧链是表面暴露的和可访问的荧光团，以使蛋白可以被标记。 <ol><li>选择残渣不是关键的，以蛋白质的功能，对于未参与配体结合位点的例子。 </li><li>在引入半胱氨酸突变，会优先考虑网站是类似的，例如丝氨酸，会导致极小扰动的蛋白质结构。 </li><li>选择标记位点后，进行突变，以确保该蛋白仅给出LRET从这些目的站点。使用标准的定点诱变方案产生突变，远离非二硫键连接的半胱氨酸可能潜在地结合成马来酰亚胺缀合荧光标记。不发生变异程二硫键连接的半胱氨酸和掩埋，游离的半胱氨酸，因为它们不应与在蛋白质的折叠形式的荧光染料反应。 </li></ol></li><li>通过使用标准的定点诱变协议点突变引入的半胱氨酸残基在所需的位点。 </li><li>包括蛋白酶切割位点可以特异性裂解掉从蛋白质中的半胱氨酸中的一个。如果蛋白质序列允许它通过保守突变蛋白质有凝血酶（识别序列LVPRGS）或引入网站Xa因子（识别序列IDGR或IEGR）附近引入的半胱氨酸序列和访问蛋白酶裂解;否则，四或六肽序列可被插入作为一个整体。选择的部位，使得在切割时所引入的半胱氨酸残基中的一个解离的其余部分蛋白在某些情况下，这可能需要两个切割位点侧翼的突变的半胱氨酸。 </li></ol> 3，测试蛋白的表达和功能<ol><li>进行免疫印迹确认突变蛋白的表达。 </li><li>执行该蛋白质的功能测定法，以保证所述突变改变了蛋白质的功能仅最低限度，如果在所有，以避免研究一种不正常的蛋白质的构象变化。 注:由于所有的蛋白质具有不同的功能，也没有一架F<html> unctional测定，是专门用于LRET;然而，功能测定法的一些实例包括酶的活性测定法的酶，配体结合测定法对受体和电生理研究离子通道。 </li></ol> 4，选择荧光基团被使用基于预期的距离范围选择荧光基团被测量，使得所述范围是0.5-1x之间的荧光团对中的R 0。 注意:这允许为背景，其典型地具有更长的寿命的一个更简单的减法。例如，如果正在测量的预期距离范围大约是35埃，一个合适的荧光团对使用将是铽螯合物作为供体和Alexa 594作为受体，因为R 0为这对是53埃（ 表1）。 5，通过瞬时转染哺乳动物细胞所需的量表达蛋白ntent"&gt;瞬时转染的兴趣到使用任何常见的转染试剂的选择哺乳动物细胞中的蛋白质通常情况下，使用每LRET实验4个10-cm的培养皿为HEK和CHO细胞系，然而，该量可以根据蛋白质表达变化性，稳定性等 ，允许细胞表达的蛋白质为收获前36-48小时。 6标签的蛋白质<ol><li>分离从培养皿的细胞。通过简单的移液缓冲对盘底部分离HEK细胞。分离的CHO细胞用细胞刮刀，洗去带外缓冲介质。 </li><li>收集细胞，离心，在1100×g离心3分钟。使用这些相同的离心分离机的设置标签后收集细胞，以及随后的洗涤之后。 </li><li>暂停细胞在3ml胞缓冲液中，然后加入等摩尔量的供体和受体荧光团高达100-300纳米的最终浓度。孵育在肩˚F或1小时，在室温下。 </li><li>洗3-4倍带外缓冲器以除去未结合的荧光团，然后暂停这些细胞在胞外缓冲液（通常为2毫升）LRET测量这些标记的细胞。 </li><li>作为对照，标签一组单独的细胞，只有供体荧光团（铽螯合物）中不添加任何受体荧光基团。 注:从这些捐助者只实验数据要完整的分析。这些实验可以在相同或不同的日子进行。 </li><li>再次，进行功能验证，此时用标记的突变体蛋白，如标号处理，也干扰功能取决于用于标记的部位。 请注意:这些实验可以在相同或不同的日子进行。 </li></ol> 7，搭建实验LRET <ol><li>将重悬的细胞中的石英比色皿与1毫升的最小体积。 </li><li>打开计算机和仪器，并调整DAT的参数à收购方案相应。 <ol><li>将激发波长为供体荧光团（330-340纳米的铽螯合物效果很好）的吸收范围。 </li><li>设置适当的发射波长，同时考虑到受体的排放变化的基础上所使用的受体。重要的是，选择尺寸仅为受体排放，不包括任何流血，通过从捐赠者的排放检测波长。例如，使用565纳米的Alexa的555波长作为受体表1。对于捐赠者只测量，其中蛋白标记只能与捐助，但没有接受，使用545纳米波长测量铽螯合物的排放。 </li><li>设定排放检测的长度至少为3倍的预期寿命LRET以确保长寿命组件将不会被错过。 </li></ol></li><li>执行扫描。做99次扫描至少三次扫描，以确保结果的一致性。除日Ë结果为文本文件（*。TXT）。 </li><li>来测量蛋白质对于不同的条件（如增加的配体）的构象变化，改变这些条件，并再次这个新的条件下对同一样品执行的每99次扫描的至少三个扫描。如果学习上的谷氨酸受体的构象谷氨酸的效果，例如，添加谷氨酸至1mM到扫描在步骤7.3中相同的样品，然后再次扫描。 </li><li>最多可添加五个单位适当的蛋白酶，并采取持续扫描，直到分裂完毕，并没有进一步的变化被认为是在生命周期的三个连续扫描。通常情况下，切割完成的两到3小时。如果Xa因子切割位点被用于蛋白酶裂解，例如，添加3μl的Xa因子的样品，并进行扫描，每30分钟3小时。 </li></ol> 8，分析得到的数据<ol><li>打开数据分析软件。 </li><li>装载荧光寿命通过输入ASCII打开文本文件中的数据。加载所有的重复以及最后的背景测量，合并成一个文件。 </li><li>平均从所有扫描的数据为各实验条件下，以得到最终的数据。要做到这一点，强调的是包含各试验中的列，然后在菜单栏中的数据菜单上单击统计上排显示从试验的平均荧光强度，以及标准差和标准差。 </li><li>积的平均值作为线图上的Y轴和寿命中的X轴，以创建表示该受体的敏化发射的寿命的曲线微秒荧光强度。改变剧情的Y轴为对数刻度，以便更好地可视化数据。 </li><li>在背景上的测量数据，重复步骤8.3，平均，显示重叠显示完全解理最终扫描。 </li><li>显示的平均背景资料相同的情节包含平均的原始数据。要做到这一点，在打开的绘图菜单中的图层控制对话框。然后，将含有背景数据传输意味着成层的内容的列表。 </li><li>对齐的平均背景数据的平均的原始数据。要做到这一点，使用在数学菜单中简单的数学函数相乘，或根据需要，直到尾部的背景与尾部的原始数据重叠划分的背景资料。 注:在此尾端，从感兴趣的蛋白质的寿命应该已经完全消失了;简单的背景信号存在什么对齐是之前和蛋白酶切割后。 </li><li>从最初的原材料LRET信号中减去对准背景信号，用简单的数学函数了。 </li><li>拟合数据以指数衰减（单个或多个指数函数，根据不同的位点和实验设计）。 <ol><li>设置起始边界拟合开始结束之后的激光脉冲。使用同样的起始点为所有后续的曲线配件。 </li><li>在选择拟合函数对话框中，选择指数衰减，以适应终身下面的等式为单一指数衰减: <img alt="式（4）" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq4.jpg" width="200" />式。 4 其中，y表示荧光强度，Y 0表示背景强度由于从系统噪声中，A 1是信号强度，t为荧光寿命，x为时间，且x 0是时间偏移量。 </li><li>修正X 0比0，开始拟合函数，并拟合数据。 注:如果实验设计只从一个对网站有信号时，所得到的数据应该很容易适合由一个单一的指数衰减8。拟合的残差是一个很好的方法来确定的拟合优度，如果额外的衰减寿命是requir主编。 </li></ol></li><li>使用从数据获得的寿命来计算使用的Forster方程（方程1和2以上的重排）的荧光团之间的距离: <img alt="式（5）" fo:content-width="2in" src="/files/ftp_upload/51895/51895eq5.jpg" width="200" />式。 5 注意:所有的变量如上面提到的与τDA已被测定为致敏受发光寿命。在R 0和R的测量进一步的细节和例子可以在其他地方7,9,10中找到。 </li><li>重复实验和数据分析至少三次，以确保重现性。同一测量个体之间的重复矛盾提出了质疑数据的有效性;使用额外的控制实验或重复。计算使用免费古斯塔夫误差分析计算器（博士托马斯·胡伯开发）或类似的系统，传播错误的测量误差适合生存期。 </li></ol>

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作者什么都没有透露。

LRET是一种强大的技术，使科学家可以在一个单一的蛋白质，以及在多聚蛋白亚基之间测量域之间的距离。正因为如此，LRET是非常适合于研究的蛋白质或其他大分子的构象变化和动力学。上述协议应允许适当装备的实验室能够轻松地测试他们的假设;然而，也有错误的可能困扰新调查许多常见来源。如果很少或没有LRET信号看出，首先检查所用的波长设置。激励应铽（330-340纳米）的吸收范围。为施主只测量，其中样品被标记只用供体荧光团和无受体荧光团的发射波长应该是在图1中示出的一个峰值，而对于施主-受主测量时，发射波长应与受体荧光团使用表1中 ，如果波长NGTH设置是否正确，检查缓冲区的兼容性。一些荧光基团可能不与某些缓冲器或在一定pH值范围内兼容。接下来，确保残留和荧光团的选择是兼容的。如果实验设计是完全正确的，那么问题可能在于无论是与荧光团或蛋白质本身。随着时间的推移，储备溶液的荧光基团的可降解，并可能导致不充分的标识。最后，检查蛋白的表达和功能。许多突变已被添加，包括引入半胱氨酸，去除天然半胱氨酸，并引进至少一种蛋白酶裂解位点。因此，可能的是，即使在正常的转染条件下，引入的突变破坏下表达目的蛋白质的蛋白质和原因，降低可见信号。如果表达的突变或标签可导致蛋白质的变性，从而使残留于被置于不同从出现在野生型蛋白质的预期距离。 Western印迹可用于验证该蛋白质的表达。如果对表面的膜蛋白的贩卖，细胞表面的生物素化和表面暴露蛋白的下拉，其次是免疫印迹感兴趣的蛋白质，将具体展示面贩卖任何问题。用于功能测试，不存在单一测定来建议使用专门与LRET，由于LRET可以在多种蛋白质类型的使用。再次，虽然，功能测定可能的例子包括酶的活性测定法，配体结合测定法，和电生理研究。如果蛋白质的表达或功能已经由引入的突变也产生不利影响，则新的标记位点必须选择。 如果一个LRET信号被看到，但不能被嵌合用单指数时有这样的结果是预期的，首先检查该背景被正确地减去。如果自动对焦之三减法，多指数衰减所看到的，这个信号可能是LRET的距离比为预期其他多重相互作用被观察的指示。检查以确保所有其他无障碍半胱氨酸已经从蛋白质中移除。如果晶体结构可用，这将是一个非常有用的工具，以检查这些半胱氨酸。再次，二硫键合和掩埋，难以接近半胱氨酸不需要将要突变的路程。为了测试这些半胱氨酸的位置偏远，如果有充分的理由不变异他们离开，引进中的蛋白质一个非天然半胱氨酸，并确保有一个供体 - 受体标记的样品中没有LRET信号。如果所有可访问的半胱氨酸确实被突变了，而且如果蛋白质具有多个亚基或者是一个复杂的一部分，那么就可能会混淆LRET信号由于染料附着于那些邻近的蛋白质或亚基。选择不同的蛋白酶裂解位点可能有助于这些问题;否则，其它标签的网站S可以需要选择。最后，如​​果该问题与嵌合仅仅是信号与噪声的问题，可能的问题是由于该蛋白的低表达。然后表达将需要通过不同的转染条件下，不同的载体等进行优化。 如果LRET测量中产生的异常或看似物理意义的结果，有可能是蛋白质特有的问题，可能无法容易地显而易见的。例如，用酸敏感离子通道，即使小心地加入一种酸来改变pH值，可能会导致一些细胞死亡和蛋白质变性。因此，多个样品需要准备，每个pH值来进行测试。另外，除了共振能量转移，本地环境的变化可以影响荧光信号。这样的改变，如果显著，将记录在捐赠者仅测量一个双重或多重指数衰减。在这些情况下，标记位点需要被移动到不同的位置，以米AKE确保在条件的变化不改变荧光团的光谱特性。 即使在保持记这些问题的来源，有一些注意事项LRET来和限制，其中一个实验者必须知道。首先，传统的标记技术依赖于标记的半胱氨酸残基。为了减少非特异性残基标签，其他半胱氨酸通常突变出来;然而，这种方法并不总是可行的。例如，如果蛋白质具有许多非二硫键连接的半胱氨酸原产于它的结构，它是对蛋白质的结构或功能是至关重要的，那么突变出来将是不可能的，大大增加了该方法的局限性和数据的解释。另外，LRET技术更适合于检测距离的变化，而 ​​不是绝对的距离，如由于取向因子κ2上的R 0值的效果上绝对距离的任何错误都可能要减少在距离变化的分析，因为这些错误会影响所有条件下进行测量平分。替代的技术可以做到克服这些局限性。例如，为了避免增加过多的半胱氨酸，人们可以贴上他的标签，并绑定到镍-NTA荧光团标签。此外，天然色氨酸可作为一个与理解的荧光团，如果用作供体，色氨酸具有小得多的寿命比铽，从而基于强度测量可以比寿命测量更合适。如果更精确的原子以原子的距离是必须的，如X射线晶体学，分子动力学，或NMR技术是更合适的技术来获得这些绝对距离。 由于其精致的灵敏度与距离的变化，LRET可以测量与埃级分辨率的距离变化为溶液相蛋白质和能提供的实验数据，而不需要高普里TY，同位素标记物或尺寸限制，即损害都NMR和分子动力学。学习和掌握技术后，检查到蛋白质的构象变化，可以更快，更容易比已有的常规方法进行。 LRET还提供了进一步的特殊共振能量转移技术，如单分子荧光共振能量转移（smFRET），它可以检查单个分子的构象状态，而不是总体平均的人口分布良好的基础。

荧光共振能量转移（LRET）是著名的荧光共振能量转移（FRET）技术1的衍生物。到FRET相似，LRET可用于测量距离和附着在上的10-100埃1-3的范围内的感兴趣的蛋白的特定位点供体和受体荧光团之间的距离的变化。是LRET的原理也类似于在该共振能量转移到FRET发生2近侧的荧光团之间时，供体荧光团的发射光谱与受体荧光团的吸收光谱重叠。这个转移的效率有关，这两个荧光团由下面的等式之间的距离: <img alt="式（1）" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq1.jpg" width="120" />式。 1 其中，R是两个荧光团之间的距离，E是连接的效率ERGY转移，且R 0，下面所讨论的，是福斯特半径为荧光团对， 即在其中传递的效率是半最大的距离。从这个等式中，可以看出，效率与上升到逆第六电源1的距离的大小。正是这种逆第六功率的依赖，允许FRET和LRET测量是连到小的距离的变化极其敏感，当FRET对的R 0附近。专门标记的蛋白质或其他大分子所需站点的能力允许我们利用这个敏感的优势，监控的构象变化。 时相比，荧光共振能量转移，其使用传统的有机染料分子，LRET提供了额外的优点。而不是使用有机染料作为供体荧光团，镧系阳离子，通常是铽或铕在LRET，用于1,4-6。那年秋天ü荧光升气管这一类， 如铽螯合物，也非常灵活的，因为它们可以具有广泛的受体荧光团的使用。这种灵活性是可能的，因为螯合的镧系元素的发射光谱包含多个尖锐的发射峰，允许一个单一物种供体荧光团能够与各种各样受体荧光团中的一个使用。因此，可以在没有任何担心从供体发射5污染泄流通过的被检测致敏的受体发射。实验者选择基于两个荧光团（ 图1和表1）之间的期望距离的特定受体。在这些螯合的镧系元素的荧光团，该金属离子是由包含敏化的通常较差吸收的镧系元素的激发以及丰富的生物反应性官能团的系绳的离子对特定官能团的高分子1的天线组分子螯合， 5,6。 ONCê兴奋，镧系元素，通过光子带衰减率在毫秒范围内释放放宽到基态。由于衰变既不是单峰对单峰松弛也不三重态到单重态放松，光子的发射不能适当地被称为荧光或磷光，而是更恰当地称为发光1。稀土发光的长期​​衰退极大地有助于寿命测量。寿命的测量可以被用来确定通过下列关系效率: <img alt="式（2）" fo:content-width="1.2in" src="/files/ftp_upload/51895/51895eq2.jpg" width="120" />式。 2 其中，E为转移的效率，τD是供体（螯合稀土）的时候不参与能量转移的寿命，τDA是供体的寿命与受体能量转移，当参与。随着LRET，τDA可以人所以被测量作为致敏受体发射的寿命，因为铽的寿命是这样比的有机受体荧光团大得多。具有相同的寿命作为其指使激发（供体的镧系元素）的受体发射，并从受自身的固有荧光寿命的寿命的任何贡献相对可以忽略不计。通过测定致敏发射，而不是供体发射，我们也无需确保标签在恰好为1:1的比例供体与受体。蛋白质可以改为同时与受体和供体荧光标记。 à不均匀标记人口造成的，但双供体标记的蛋白质将不会发出在接受波长和双受体标记的蛋白质将不激动。此外，荧光团之间的距离应该是相同的，而不管其中的半胱氨酸位点的特定荧光团附着，使用各向同性的镧系元素作为供体尤其是当，所以娘家姓d，来指定一个特定的网站分别接受供体或受体是不必要的。强度可能会受到与异质群体，但仍应该超过足以被检测到。 在规划的实验中，荧光团的选择应是R 0值的一对，以及预期的距离范围来规定被测量。在R 0的值被定义由下面的等式: <img alt="式（3）" fo:content-width="2.5in" src="/files/ftp_upload/51895/51895eq3.jpg" width="250" />式。 3 其中，R 0为福斯特半径埃，κ2是两种染料（通常假设为三分之二）之间的取向因子，φD为供体的量子产率，J为光谱重叠积分捐赠者的间发射光谱和受体的吸收光谱中的M - 1-1处4，且n是介质1的折射率。 我们的实验室已通过对蛋白质的供体和受体标记位点之间引入蛋白酶识别位点被探测添加修改常规LRET技术。该变形允许调查在未纯化系统，例如整个哺乳动物细胞7。使用半胱氨酸作为位点标记时，由于在与结合于半胱氨酸的巯基，其它蛋白质上具有半胱氨酸还标记了的细胞的马来酰亚胺共轭染料标记的过程中该技术是特别有用的。然而，通过包括对感兴趣的蛋白的蛋白酶裂解位点和之前和裂解后测量的寿命，实验者可以定量地减去从原始信号蛋白酶裂解后的背景信号。这个减法隔离感兴趣的蛋白质产生的特定信号（ 图尤尔2）。使用上述的变形例中，LRET可用于测量铽螯合物供体和对蛋白质的受体探针之间的距离的变化，并由此监视未经纯化的要求，在蛋白质的接近生理状态的构象变化。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig1highres.jpg" width="500" /> 图1，吸收和螯合铽在黑，以及作为代表性受体的Alexa 488，在红色的发射光谱图。注意，多个发射峰和铽螯合物的各峰的尖锐，窄的发射范围。这种模式允许对与多种受体荧光团的用铽和便于这些范围，其中铽表明没有发射内致敏发射的测量。在486铽的发光峰值波长与A重叠相当不错<html>的Alexa 488的bsorption峰，允许共振能量转移两个荧光团之间发生。 515nm的波长是一个很好的选择，检测致敏排放为这双，因为它是在铽发射峰之间的山谷中，相当接近520纳米的Alexa 488的发射峰。注意，被附近的受体峰，虽然理想的，则不需要-565（nm）是仍然能够检测到没有同时检测铽发射的Alexa 488发射。 <table border="0" cellpadding="0" cellspacing="0" width="374"><tbody><tr height="27"><td height="27" style="height:27px;width:147px;"> 受体荧光基团 </td><td style="width:45px;"> R 0（） </td><td style="width:183px;"> 发射波长（nm） </td></tr><tr height="23"><td height="23" style="height:23px;width:147px;">阿托465 </td><td align="right" style="width:45px;"> 36 </td>3px的;"&gt; 508 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;">荧光素</td><td align="right" style="width:45px;"> 45 </td><td align="right" style="width:183px;"> 515 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的488 </td><td align="right" style="width:45px;"> 46 </td><td align="right" style="width:183px;"> 515 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的680 </td><td align="right" style="width:45px;"> 52 </td><td align="right" style="width:183px;"> 700 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的594 </td><td align="right" style="width:45px;"> 53 </td><td align="right" style="width:183px;"> 630 </td></tr><tr height="22"><td height="22" style="height:22px;width:147px;"> Alexa的555 </td><td align="right" style="width:45px;"> 65 </td><td align="right" style="width:183px;"> 565 </td></tr><tr高度="22"&gt; <td height="22" style="height:22px;width:147px;"> Cy3标记</td><td align="right" style="width:45px;"> 65 </td><td align="right" style="width:183px;"> 575 </td></tr></tbody></table> 表1常用的受体荧光基团用铽螯合物作为捐助11 LRET列表。在R 0的值进行测定时，供体和受体被装AMPA受体的可溶性受体激动剂结合域。它是理想的，重新测量R 0值正在研究每一个新的系统。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig2highres.jpg" width="500" /> 图2呈现的LRET方法的概述。（A）中的 AMPA受体是一种膜蛋白，其经历在配体结合构象变化。在翻盖型配体的双nding域在这里红色圆圈。 （B）存在于开放构象（左）AMPA的在未结合蛋白的配体结合结构域。当绑定到配体谷氨酸，在蛋白质周围的配体（右）关闭。通过将荧光团在证明的站点上的发光二极管，此构象变化的性质，可以被看作是荧光团的改变，这将影响随后的荧光寿命之间的距离。 （C）当标记的全细胞，可能会发生的利息两者蛋白质以及背景的膜蛋白的标记（左）。蛋白酶裂解后，从感兴趣的蛋白质LRET信号就会消失，由于可溶性片段的释放，而使背景信号完好（右）。此背景信号然后可减去从原始信号。

<table><tbody><tr><td>LanthaScreen Thiol Reactive Tb Chelate</td><td>Life Technologies</td><td>PV3579</td><td></td></tr><tr><td>Acceptor fluorophore-Fluorescein-5-Maleimide</td><td>Life Technologies&amp;nbsp;</td><td>F-150</td><td>Choice of acceptor depends on the specific experiment</td></tr><tr><td>Acceptor fluorophore-Alexa Fluor 488 C5 Maleimide</td><td>Life Technologies&amp;nbsp;</td><td>A-10254</td><td>Choice of acceptor depends on the specific experiment</td></tr><tr><td>Acceptor fluorophore-Alexa Fluor 555 C2 Maleimide</td><td>Life Technologies&amp;nbsp;</td><td>A-20346</td><td>Choice of acceptor depends on the specific experiment</td></tr><tr><td>Acceptor fluorophore-Alexa Fluor 594 C5 Maleimide</td><td>Life Technologies&amp;nbsp;</td><td>A-10256</td><td>Choice of acceptor depends on the specific experiment</td></tr><tr><td>Acceptor fluorophore-Alexa Fluor 680 C2 Maleimide</td><td>Life Technologies&amp;nbsp;</td><td>A-20344</td><td>Choice of acceptor depends on the specific experiment</td></tr><tr><td>QuantaMaster 3-SS</td><td>Photon Technology International</td><td></td><td>Spectrofluorometer should have pulsed excitation with the ability to measure lifetimes in the millisecond range</td></tr><tr><td>FluoreScan 2.0</td><td>Photon Technology International</td><td></td><td>Data Acquisition Software used in manuscript. Software provided with fluorescence instrument</td></tr><tr><td>Origin 8.6</td><td>OriginLab</td><td></td><td>Origin is the data analysis software used in protocol. Can use other similar data analysis software</td></tr><tr><td>Quartz cuvette</td><td>Starna Cells, Inc</td><td>3-Q-10</td><td></td></tr><tr><td>Stir bar&amp;nbsp;</td><td>Bel-Art Products</td><td>F37119-0007</td><td>Used in cuvette to keep cells in suspension. Can use any stir bar that fits the cuvette</td></tr></tbody></table>

luminescence resonance energy transfer to study conformational changes in membrane proteins expressed in mammalian cells

发光共振能量转移，或LRET，是用来测量在10-100埃的距离范围内的蛋白质两个站点之间的距离的功能强大的技术。通过测量各个连接的情况下的距离，该蛋白质的构象变化可以很容易地进行评估。与LRET，镧系元素，最多螯合铽，用作供体荧光团，得到的优点，​​如更长的供体 - 仅发光寿命，使用多个受体荧光团的灵活性，并有机会检测致敏受体发射作为一种简单的方法测量不也检测供体只有信号的风险能量转移。在这里，我们描述了使用LRET上表达，并测定完整的哺乳动物细胞的表面上的膜蛋白的方法。我们介绍了荧光LRET对之间蛋白酶切割位点。获取原始LRET信号后，乳沟在该网站将删除的蛋白的特异性信号LRET息使我们能够定量地减去该保持切割后​​的背景信号。此方法允许更要作出生理学相关的测量，而不需要蛋白质的纯化。

一个成功的LRET测量与镧系捐赠者应该有一个捐赠者只在毫秒级的时间范围内的寿命。致敏LRET排放的标有两个供体和受体的蛋白质的寿命会明显缩短，与后背景减除图3微秒范围内的寿命。蛋白酶裂解导致增加的寿命应该成为稳定一段时间（ 即不再改变），这表明蛋白酶切割完成图4，如果LRET信号来自只有一组位点，背景减去后所得到的发光寿命应该给单指数寿命。 当测量的构象变化，具体LRET信号应显示在寿命的测量图3中的错误之外 ​​的变化。可以由误差的传播来计算测量的误差S采用了寿命的配合有关。拟合数据的方程4，寿命，τ描述指数式衰减函数的最小数目后，确定即可。用式（5）中，供体-受体和供体-仅寿命可以随着R 0值用于LRET一对来计算在测试条件下这两个荧光团之间的距离。有关从改变这些条件对蛋白质的总体结构和功能产生的距离的变化是现在实验者的作业。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig3highres.jpg" width="500" /> 图3 LRET测量酸敏感离子通道（ASIC1a）的 。荧光强度已经被绘制在对数刻度，以提高易用性的视觉诠释。 （ 一）捐赠者只样本显示单exponentia升衰变不随pH值变化。 （b）在给体-受体标记的样品，致敏排放的寿命减少被认为是在pH值下降8〜6（黑红色）。这个寿命降低表明在ASIC1a的手指和拇指的域之间的距离的减小。这个数字已经被修改拉马斯瓦米等 ，2013 8。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/51895/51895fig4highres.jpg" width="500" /> 图4的背景减除上作出对酸敏感离子通道（ASIC1a）LRET测量的影响。的网站，以进行具体的荧光团通过蛋白酶切割位点被分离的标签。后LRET测量时，所述蛋白酶被引入到蛋白质样品与感​​兴趣的结果蛋白质的后续切割，在特定信号的损失。剩下的任何LRET是从荧光团结合到存在于其它膜蛋白的其他半胱氨酸背景荧光。减去该背景分离的真LRET数据对感兴趣的蛋白质。这个数字已经被修改拉马斯瓦米等 ，2013 8。

Watch this Scientific Journal Video about Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells at JoVE.com

Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

我们在这里介绍了一种改进荧光共振能量转移（LRET）方法，在这里我们介绍的供体和受体荧光基团的站点之间的蛋白酶裂解位点。该变形使得我们能够获得来自感兴趣膜蛋白，使膜蛋白的无蛋白质纯化的研究中出现的具体LRET信号。

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

Luminescence Resonance Energy Transfer, or LRET, is a powerful technique used to measure distances between two sites in proteins within the distance range ...

luminescence-resonance-energy-transfer-to-study-conformational

Nanyang Technological University

Research

JoVE Journal

Bioengineering

12.0K 次观看.  University of Texas Health Science Center at Houston. 我们在这里介绍了一种改进荧光共振能量转移（LRET）方法，在这里我们介绍的供体和受体荧光基团的站点之间的蛋白酶裂解位点。该变形使得我们能够获得来自感兴趣膜蛋白，使膜蛋白的无蛋白质纯化的研究中出现的具体LRET信号。 

视频: Luminescence Resonance Energy Transfer to Study Conformational Changes in Membrane Proteins Expressed in Mammalian Cells

Using In Vitro Fluorescence Resonance Energy Transfer to Study the Dynamics Of Protein Complexes at a Millisecond Time Scale

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological functions. Such interactions can be highly dynamic, meaning the interacting subunits are constantly associated and dissociated at certain rates. While measuring the binding affinity using techniques such as quantitative pull-down reveals the strength of the interaction, studying the binding kinetics provides insights on how fast the interaction occurs and how long each complex can exist. Furthermore, measuring the kinetics of an interaction in the presence of an additional factor, such as a protein exchange factor or a drug, helps reveal the mechanism by which the interaction is regulated by the other factor, providing important knowledge for the advancement of biological and medical research. Here, we describe a protocol for measuring the binding kinetics of a protein complex that has a high intrinsic association rate and can be dissociated quickly by another protein. The method uses fluorescence resonance energy transfer to report the formation of the protein complex in vitro, and it enables monitoring the fast association and dissociation of the complex in real time on a stopped-flow fluorimeter. Using this assay, the association and dissociation rate constants of the protein complex are quantified.

Protein-protein interactions are critical for biological systems, and studies of the binding kinetics provide insights into the dynamics and function of protein complexes. We describe a method that quantifies the kinetic parameters of a protein complex using fluorescence resonance energy transfer and the stopped-flow technique. &#160;

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

Proteins are the primary operators of biological systems, and they usually interact with other macro- or small molecules to carry out their biological ...

科研

生物化学

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

单分子荧光能量转移研究核糖体蛋白质合成

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques. In dual-color FCCS, where the fluctuations in fluorescence intensity represent the dynamic &#34;fingerprint&#34; of the respective fluorescent biomolecule, we can probe co-diffusion or binding of the receptors. FRET, with its high sensitivity to molecular distances, serves as a well-known &#34;nanoruler&#34; to monitor intramolecular changes. Taken together, conformational changes and key parameters such as local receptor concentrations and mobility constants become accessible in cellular settings.
Quantitative fluorescence approaches are challenging in cells due to high noise levels and the vulnerability of the sample. Here we show how to perform this experiment, including the calibration steps using dual-color labeled &#946;2-adrenergic receptor (&#946;2AR) labeled with eGFP and SNAP-tag-TAMRA. A step-by-step data analysis procedure is provided using open-source software and templates that are easy to customize.
Our guideline enables researchers to unravel molecular interactions of biomolecules in live cells in situ with high reliability despite the limited signal-to-noise levels in live cell experiments. The operational window of FRET and particularly FCCS at low concentrations allows quantitative analysis at near-physiological conditions.

We present an experimental protocol and data analysis workflow to perform live cell dual-color fluorescence cross correlation spectroscopy (FCCS) combined with F&#246;rster Resonance Energy transfer (FRET) to study membrane receptor dynamics in live cells using modern fluorescence labeling techniques.

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

We present a protocol and workflow to perform live cell dual-color fluorescence cross-correlation spectroscopy (FCCS) combined with F&#246;rster Resonance ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

福斯特共振能量转移映射:阐明全球结构特征的新方法

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the environment, a cell must be able to recognize and process it. This task mainly relies on the function of membrane receptors, whose role is to convert signals into the biochemical language of the cell. G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptor proteins in humans. Among GPCRs, metabotropic glutamate receptors (mGluRs) are a unique subclass that function as obligate dimers and possess a large extracellular domain that contains the ligand-binding site. Recent advances in structural studies of mGluRs have improved the understanding of their activation process. However, the propagation of large-scale conformational changes through mGluRs during activation and modulation is poorly understood. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to visualize and quantify the structural dynamics of biomolecules at the single-protein level. To visualize the dynamic process of mGluR2 activation, fluorescent conformational sensors based on unnatural amino acid (UAA) incorporation were developed that allowed site-specific protein labeling without perturbation of the native structure of receptors. The protocol described here explains how to perform these experiments, including the novel UAA labeling approach, sample preparation, and smFRET data acquisition and analysis. These strategies are generalizable and can be extended to investigate the conformational dynamics of a variety of membrane proteins.

This study presents a detailed procedure to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments on G protein-coupled receptors (GPCRs) using site-specific labeling via unnatural amino acid (UAA) incorporation. The protocol provides a step-by-step guide for smFRET sample preparation, experiments, and data analysis.

使用单分子FRET可视化膜受体的构象动力学

The ability of cells to respond to external signals is essential for cellular development, growth, and survival. To respond to a signal from the ...

使用基于 BRET 的方法在活细胞中进行 CRAF-14-3-3 相互作用分析

Bioluminescence Resonance Energy Transfer (BRET)-Based Assay for Measuring Interactions of CRAF with 14-3-3 Proteins in Live Cells

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot for germline mutations, which are shown to cause the developmental RASopathy, Noonan syndrome. All RAF kinases contain multiple phosphorylation-dependent binding sites for 14-3-3 regulatory proteins. The differential binding of 14-3-3 to these sites plays essential roles in the formation of active RAF dimers at the plasma membrane under signaling conditions and in maintaining RAF autoinhibition under quiescent conditions. Understanding how these interactions are regulated and how they can be modulated is critical for identifying new therapeutic approaches that target RAF function. Here, I describe a bioluminescence resonance energy transfer (BRET)-based assay for measuring the interactions of CRAF with 14-3-3 proteins in live cells. Specifically, this assay measures the interactions of CRAF fused to a Nano luciferase donor and 14-3-3 fused to a Halo tag acceptor, where the interaction of RAF and 14-3-3 results in donor-to-acceptor energy transfer and the generation of the BRET signal. The protocol further shows that this signal can be disrupted by mutations shown to prevent 14-3-3 binding to each of its high-affinity RAF docking sites. This protocol describes the procedures for seeding, transfecting, and replating the cells, along with detailed instructions for reading BRET emissions, performing data analysis, and confirming protein expression levels. In addition, example assay results, along with optimization and troubleshooting steps, are provided.

作者聚焦：整合基于 BRET 的检测和罕见突变分析以破译活细胞中的 RAF 激酶调控

This protocol describes a BRET-based assay for measuring the interactions of the CRAF kinase with 14-3-3 proteins in live cells. The protocol outlines steps for preparing the cells, reading BRET emissions, and data analysis. An example result with identification of appropriate controls and troubleshooting for assay optimization is also presented.

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

CRAF is a primary effector of RAS GTPases and plays a critical role in the tumorigenesis of several KRAS-driven cancers. In addition, CRAF is a hotspot ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

生物学

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

内质网子域的培养细胞中的可视化

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

摘要

探索更多视频

此视频中的章节

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

单分子荧光能量转移研究核糖体蛋白质合成

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

福斯特共振能量转移映射:阐明全球结构特征的新方法

使用单分子FRET可视化膜受体的构象动力学

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

研究膜蛋白的构象动力学原位网站的荧光标记

内质网子域的培养细胞中的可视化

荧光共振能量转移研究构象变化的膜蛋白在哺乳动物细胞中表达

摘要

探索更多视频

此视频中的章节

利用体外荧光共振能量转移研究毫秒时间尺度蛋白质配合物的动力学

单分子荧光能量转移研究核糖体蛋白质合成

双色荧光互相关光谱研究活细胞中蛋白质-蛋白质相互作用和蛋白质动力学

福斯特共振能量转移映射:阐明全球结构特征的新方法

使用单分子FRET可视化膜受体的构象动力学

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移 （BRET） 的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

研究膜蛋白的构象动力学原位网站的荧光标记

内质网子域的培养细胞中的可视化

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用

基于生物发光共振能量转移（BRET）的测定法，用于测量 CRAF 与活细胞中 14-3-3 蛋白的相互作用