Name: identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling
Uploaded: 2016-09-20T08:00:00.000+00:00
Duration: 10 min 49 s
Description: Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

We thank Dr. Ben Nacev for advice on design of the photoaffinity labeling protocol, Dr. Wei Shi for synthesizing the itraconazole photoaffinity probe, Dr. Yongjun Dang for advice on affinity pull-down experiments, and other members of the J.O.L. laboratory for helpful comments and support. This work was supported in part by a PhRMA Foundation Fellowship in Pharmacology/Toxicology (to S.A.H.); National Cancer Institute Grant R01CA184103; the Flight Attendant Medical Research Institute; Prostate Cancer Foundation (J.O.L.); and the Johns Hopkins Institute for Clinical and Translational Research, which is funded in part by Grant UL1 TR 001079 from the National Center for Advancing Translational Sciences (NCATS).

注:此协议是改编自麦金农和汤顿10在活细胞中的使用。 1.培养细胞的制备<ol><li>制备无菌6厘米细胞培养皿为所需的样本数（见下文）。 注:细胞的一个培养皿每个处理条件下使用，但如果需要更多的蛋白2或3菜肴每种条件进行制备和UV照射后组合。 <ol><li>制备的细胞中的至少3菜肴;阴性对照与DMSO只（D）中，只有探头治疗（P）和探头+竞争对手（C）。 </li><li>任选地，准备4 个菜作为无紫外线控制，与探针进行处理，但不进行UV光。 注意:如果多个竞争者分子进行测试，如感兴趣的小分子的不同的类似物，制备额外竞争者板是必要的。 </li></ol></li><li> HEK293T细胞添加到培养皿在4毫升培养基中。使用350万Ç每盘厄尔。 注:应确定细胞的数目对于每个细胞类型，使得细胞在实验开始时几乎100％汇合，以达到最大的蛋白质产量。对于HUVEC，用每盘30万细胞。一个好的近似是一个融合的15厘米菜细胞的1/10。 HEK293T细胞应该在被培养的Dulbecco氏改良的Eagles培养基补充有10％胎牛血清（FBS）和1％青霉素/链霉素。 </li><li>返回细胞培养培养箱中（37℃，5％CO 2）过夜。 </li></ol> 2.细胞和光交联处理<ol><li>第二天，预等分下列药物入1.5ml微量管: <ol><li>对于第一次治疗，加入4微升DMSO中至2管和4微升10毫竞争者（即未改性的母体化合物）的1管。 </li><li>用于第二处理中，添加16微升的DMSO以1管和16微升的50＃181，M光亲探至2管。 注:最佳浓度可能是不同的探针（见讨论）不同，但在200-500纳米之间，是一个很好的起点（这里我们使用200纳米）。竞争者的浓度应超过使探针至少20倍（这里我们使用10μM）的。 DMSO的终浓度应在所有平板相等，并应不超过0.5％（20微升体积）。 </li></ol></li><li>使细胞板到细胞培养罩并且从步骤2.1.1添加预先等分的药物如下:吸1毫升培养基，再悬浮预等分药物在媒体上，并轻轻地加入它回到板降明智的。二甲基亚砜加入到二甲基亚砜（D）的板和探针（P）的板，和竞争添加到竞争（C）的板。返回所述板的培养孵化30分钟。 </li><li>在调暗培养罩灯，从步骤2.1.2添加-等分预探针来探测（P）和竞争（C）的板，并添加DMSO中的二甲基亚砜（D）的板，如上述。返回所述板的培养孵化1小时。 </li><li>在培育期间，请准备以下物品;冰托盘可以容纳所有的板块;冰冷的裂解缓冲液（PBS [pH为8.5] + 1×蛋白酶抑制剂鸡尾酒[完整无EDTA，根据水中的50倍原液稀释]; 200微升/板）;离心管标记为D，P或C的每个的不同的处理条件下，在冰上。 </li><li> 1小时培养结束前15分钟，成立了紫外灯（365纳米），在寒冷的房间，并打开它热身灯泡。 </li><li>与探针孵育1小时后，放置碗碟冰上。清洗细胞轻轻用5ml冰冷的PBS（pH 7.4）中以除去过量的探针。再覆盖用4ml冰冷的PBS中的细胞。 </li><li>将集中在冰袋上方的紫外灯在3厘米至最小化从灯加热单元的菜。照射3分钟。取下菜冰托盘并重复所有样品。 </li><li>一个压脚提升照射，从细胞吸出PBS并添加冰冷的PBS（pH为8.5）与蛋白酶抑制剂对各板的200微升。从使用橡胶刮刀，并转移到冰上预标记的微量离心管板分离细胞。 </li><li>加入SDS至0.4％的终浓度（10微升10％SDS的入250微升样品的）。 注意:SDS粉是危险的。避免穿了鼻子和嘴口罩吸入SDS粉末。 </li><li>裂解的细胞通过超声处理该悬浮液为10个脉冲（输出1，占空比30％）和第二轮10的脉冲之前在冰上孵育1分钟。 </li><li>如果需要的话，除去2微升悬浮液并在光学显微镜下检查，以确保完全细胞裂解。 </li><li>沸腾的热板上设置为95℃5分钟，以完成细胞裂解和变性的所有蛋白质的样品。 </li><li>使用分光光度洗涤剂相容蛋白测定中，如第测定各样品中的蛋白质浓度ÈDC蛋白测定19，根据制造商的说明，用设定在750纳米测量吸光度分光光度计。 </li><li>通过根据需要加入的PBS pH值8.5±0.4％SDS的正常化蛋白质浓度2.5毫克/毫升（或如果浓度低于2.5毫克/毫升，正常化到具有最低蛋白质浓度样品）（例如，如果样品是5在250微升毫克/毫升，加入250微升的PBS pH 8.5的0.4％的SDS，得到2.5毫克/毫升终浓度）。 </li></ol> 3.附件荧光标签通过点击化学的标记蛋白的可视化<ol><li>除去将40μl细胞裂解物并转移到一个新的离心管中。保持其余裂解物与生物素 - 叠氮化物（步骤4）的反应。 </li><li>添加下列试剂依次是:0.2微升氟叠氮（1毫米原液在DMSO），0.58微升TCEP（100毫米股票在水中4当量的NaOH加入，新鲜制备）和3.38微升TBTA（1。在4 7毫米股票:叔丁醇到DMSO 1的比例）。旋涡混合。 </li><li>添加1.14微升的CuSO 4 -5H 2 O（50毫在水中）以开始反应。短暂涡旋并孵育在室温下在黑暗中30分钟。 </li><li>加入50μl2×SDS样品缓冲液以终止反应。此时，直接在SDS-PAGE凝胶上20运行样品最好的结果，或在-20℃过夜如果需要存储。 </li><li>如果冷冻样品装载到凝胶之前，在95℃再加热5分钟。 注:由于目标蛋白质的分子量通常在此阶段不公知的，最好是运行2凝胶具有不同的丙烯酰胺浓度（ 即 ，8％和15％），或宽分子量范围的梯度凝胶（即 4-15％），以确保完全分子量覆盖。 </li><li>当染料前沿到达凝胶的末端，继续运行凝胶额外5分钟，以确保所有的过量unreacte的ð氟叠氮化合物已经完全退出了凝胶。 </li><li>与DDH 2 O的取出凝胶的容器和孵育10分钟，轻轻摇动，洗去所有多余的氟，叠氮化物。 </li><li>将凝胶在玻璃板上，并使用根据制造商的说明一台风荧光凝胶扫描仪扫描凝胶。 </li></ol> 4.附生物素标记的通过点击化学的标记蛋白的亲和纯化<ol><li>从步骤3.1剩余裂解物，使用的最大量的蛋白质归一化之后，使得所有的样品是相同的体积（例如，如果标准化后到2.5毫克/毫升所得的样品体积是500，550，和600微升，使用500每微升）。 </li><li>通过加入50微升的高容量链霉琼脂糖珠，预洗涤2次，用PBS（pH 7.4）中预清除裂解物。孵育1小时在4℃轮换。 </li><li>沉淀通过离心珠在1000 xg离心3分钟。除去叔他上清液在冰上一个新的离心管并弃去珠。 </li><li>除去DMSO样品1-2％到标有"输入"的新管中。添加2×SDS样品缓冲液并储存的等效体积在-20℃。 </li><li>每500微升裂解液，添加下列试剂:1.38微升生物素 - 叠氮化物（10毫米股票DMSO），5.5微升TCEP，和32.5微升TBTA。旋涡混合。 </li><li>加入11微升硫酸铜每500微升裂解液和旋涡简要-5H 2 O。在室温下孵育30分钟。 </li><li>加冷却到-20℃的丙酮4样品体积。涡旋样品，并在-80℃孵育过夜以完全沉淀的蛋白质和除去未反应的生物素 - 叠氮化物。 </li><li>离心机在17000×g离心15分钟，在4℃下将样品以沉淀沉淀的蛋白质。 </li><li>完全吸出上清液，并在150μl的PBS（pH为7.4）+ 1％SDS通过超声处理resolubilize的蛋白质。 </li><li&gt;添加600μl的PBS（pH 7.4）中，以SDS浓度稀释至0.2％。 </li><li>样品添加到30微升洗涤的预高容量链霉琼脂糖珠，并在4℃下孵育1小时以转动。 </li><li>沉淀通过离心珠在1000 xg离心3分钟。吸出含有未结合的蛋白，弃上清。 </li><li> 1毫升洗涤缓冲液（400毫摩尔NaCl，50毫摩尔Tris，0.2％SDS，pH7.4）中添加到珠子和孵化在室温下用旋转5分钟。 </li><li>重复步骤4.12和4.13的3倍。 </li><li>从珠粒完全吸出洗涤缓冲液，并加入30微升2×SDS样品缓冲液中。 </li><li>孵育5分钟95℃的加热块上，以从珠释放的蛋白质。 </li><li>离心1分钟的珠粒在室温下以13000 xg离心。 注意:在这一点上，可将样品储存在-20℃，直至准备好运行的SDS-PAGE。如果冷冻样品，在使用前在95℃下再加热5分钟。 </li><li>小心吸取了含有的蛋白质珠样品缓冲液，并加载到SDS-PAGE 20（见下文重要的考虑因素）。珠可以保存如有必要，以后再沸腾。 注:对于由银染色蛋白质检测，使用1mm厚的凝胶，得到更好的结果。如果频带要被切出质谱（MS）分析的银染凝胶，制备从无菌溶液新鲜的所有试剂，并在凝胶上采样之间留下一个空孔。为从探头车道切割每个频带，一个平行切片应从二甲基亚砜车道可采取使背景蛋白可以减去。对于通过蛋白质印迹蛋白的ID验证，这是至关重要的运行SDS-PAGE凝胶时也从步骤4.4加载的输入采样。 </li><li>遵循两种银染21或印迹22检测靶蛋白（S）的标准协议。 </li></ol>

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R., Shifrin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+highly+sensitive+silver+stain+for+detecting+proteins+and+peptides+in+polyacrylamide+gels.">A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels.</a> Anal Biochem. 98 (1), 231-237 (1979).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> P Natl Acad Sci USA. 76 (9), 4350-4354 (1979).</li></ol>

The authors have nothing to disclose.

不同的方法来鉴定小分子的目标，大致可分为两类:自上而下，其中药物的细胞表型被用于基于其已知功能来缩小其潜在的目标，或自下而上，其中目标通过化学或遗传方法3直接识别。自上而下或表型的研究可以识别受药物的某些细胞过程（ 例如 ，转录/翻译/ DNA合成，细胞周期阻滞，信号通路的激活/禁止等 ）可能背后的小分子的最终表型，这帮助的潜在目标的列表缩小到?...

生物活性的小分子基本上通过与相互作用并改变一个或多个"目标"的分子，最常见的蛋白质的功能，在小区工作。在药物发现中，当活性化合物是通过表型筛选发现，该化合物的分子靶（多个）的识别是关键的，不只是对于理解动作和化合物的潜在副作用的机制，但也潜在发现新的生物学基础的疾病模型，从而为新机制类治疗1发展道路。虽然不要求目标识别的一种药物用于治疗，近年来出现了越来越认识到，新的候选药物更容易在临床试验中取得成功，并因此产生更好的投资回报，如果一个有效的目标是已知2。因此，一直存在的方法用于鉴定小的兴趣日益增​​长分子靶蛋白。 一个经典的目标识别试验通常依赖于亲和纯化，其中感兴趣的小分子被固定在树脂并用全细胞裂解物，在这之后未结合的蛋白质被冲走，而其余的蛋白质被洗脱，并确定3温育。虽然这一技术已被用来鉴别许多小分子4的目标，它不适合作为用于几个原因的通用目标ID方法。首先，目标蛋白质必须保留在细胞裂解其天然构象，以保留其结合到小分子的能力。这可以是膜蛋白，其通常经历构象变化从它们的天然环境中取出后，或简单地聚合和溶液中沉淀出来尤其成问题。第二，小分子必须用化学方法以这样的方式，它可以固定到树脂改性，同时保持其与靶蛋白结合的能力。一旦被固定于树脂深结合口袋可能因此变得不可访问的小分子。第三，结合亲和力必须足够高，使得在洗涤步骤的相互作用被维持，使得低亲和力相互作用有挑战性的鉴定。如pH值，离子浓度，或其他内源性分子的存在第四，环境条件可在细胞内空间变化，并且有时会发生药物 - 靶相互作用的先决条件。因此，找到正确的条件，以允许和维持结合细胞外可能需要反复试验的一个显著量。 光亲和标记通过允许共价小分子和细胞的天然环境中的目标的结合规避这些问题。而不是固定的小分子要大笨重的树脂，在分子，而不是化学修饰安装两个小功能摹roups:一个光活化部分，其允许共价交联到靶蛋白时的光的特定波长照射，和一个报告基团，它允许待检测的靶蛋白，并随后分离。活细胞用的光亲和探针处理，探针结合和共价交联到靶蛋白，和探针 - 蛋白质复合物，然后分离完好。探针结合到靶的特异性由并联，其中过​​量的母体化合物的用于竞争远到靶蛋白的探针的结合进行竞争实验证明。 光亲和探针的设计和合成变化很大从一个小分子到另一个，并且不会在这个协议中支付;然而，关于这个问题的几个优秀的讨论已经发表5-9。主要考虑的是，该探针保留母体化合物的生物活性，因此presumabLY结合相同靶（多个）。结构 - 活性关系（SAR）研究必须进行，以确定其中分子的部分可以在不活性的损失进行修改。多种不同化学基团的已被用作可光活化交联剂，包括diazirine，二苯甲酮和芳基叠氮化物，它们各自具有优点和缺点10。同样地，也有已经用于分离探针结合蛋白多记者标记。报告基团可以是对自己的，功能如常用的生物素或荧光标记，或可能是需要的光交联步骤，其中有被小，因此不太可能损害生物活性11的优点随后进一步官能的前体。 在这个协议中，我们已经使用含有diazirine光致团的光亲探针，以及用于报告基团的通过的Cu（Ⅰ）的附接末端炔-catalyzED叠氮炔夏普勒斯-胡伊斯根环（或点击）反应12-15。特区研究，探针的设计和合成，以及这些研究的结果已被别处16-18出版。

<table><tbody><tr><td>Tris(2-carboxyethyl)phosphine (TCEP)</td><td>Life Technologies</td><td>20490</td><td>Make fresh day of use. Prepare 100 mM stock in water with 4 eq NaOH.</td></tr><tr><td>Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] amine (TBTA)</td><td>AnaSpec</td><td>63360-50&amp;nbsp;</td><td>Prepare 1.7 mM stock in a 4:1 ratio of t-butanol to DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Copper Sulfate (CuSO&lt;sub&gt;4&lt;/sub&gt;&amp;#8226;5H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>LabChem, Inc.</td><td>LC13440-1</td><td>Prepare 50 mM stock in water, store at room temperature.</td></tr><tr><td>Biotin-azide</td><td>Click Chemistry Tools</td><td>AZ104-100</td><td>Prepare 10 mM stock in DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Alexa Fluor 647-azide&amp;nbsp;</td><td>Life Technologies</td><td>A10277</td><td>Prepare 1 mM stock in DMSO, store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>365 nm UV lamp</td><td>Spectroline</td><td>FC100</td><td>UV-blocking glasses should be worn while operating.</td></tr><tr><td>Protease inhibitor tablets, EDTA-free</td><td>Roche Life Science</td><td>11873580001</td><td>Prepare 50x solution in water and store at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Sonicator</td><td>Branson</td><td>Sonifier 250</td><td>Set to output 1, duty 30%.&amp;nbsp;</td></tr><tr><td>Fluorescent gel scanner</td><td>GE Healthcare Life Sciences</td><td>FLA 9500</td><td>Use red laser to detect Alexa-fluor 647.</td></tr><tr><td>Detergent-compatible Dc protein assay kit</td><td>Bio-Rad</td><td>5000112</td><td></td></tr><tr><td>High Capacity Streptavidin Agarose beads&amp;nbsp;</td><td>Life Technologies</td><td>20359</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagles Medium, low glucose</td><td>ThermoFisher Scientific</td><td>11885092</td><td></td></tr><tr><td>Fetal Bovine Serum, qualified</td><td>ThermoFisher Scientific</td><td>26140079</td><td></td></tr><tr><td>Penicillin/Streptomycin solution</td><td>ThermoFisher Scientific</td><td>15140122</td><td></td></tr><tr><td>SDS Sample Buffer (2x)</td><td>ThermoFisher Scientific</td><td>LC2676</td><td></td></tr></tbody></table>

identification of small molecule-binding proteins in a native cellular environment by live-cell photoaffinity labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

这里示出的结果与抗真菌药伊曲康唑，它的使用，已经出版16的光亲和性探针获得。这些结果表明使用活细胞光亲和标记技术在成功地识别的主要依他康唑结合蛋白作为35kDa的膜蛋白电压依赖性阴离子通道1（VDAC1）。 上述方案是使用用于photolabeling和未修饰的伊曲康唑作为竞争分子的依他康唑的探针在HEK293T细胞中进行。...

Watch this Scientific Journal Video about Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling at JoVE.com

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

小分子结合蛋白的鉴定在天然细胞环境由活细胞光亲和标记

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

identification-small-molecule-binding-proteins-native-cellular

Research

JoVE Journal

Biochemistry

12.6K 次观看.  Johns Hopkins University School of Medicine. We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis. 

视频: Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

检测和可视化DNA损伤诱导的蛋白质复合物悬浮细胞培养使用邻近连接测定

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

科研

生物化学

Split-BioID &#8212; Proteomic Analysis of Context-specific Protein Complexes in Their Native Cellular Environment

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells. One such enzyme, BirA* (used in the BioID approach), mediates the biotinylation of proteins within a range of approximately 10 nm. Hence, when fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment. As opposed to AP that relies on the purification of assembled protein complexes, BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated. Since it biotinylates proximal proteins, one can moreover capitalize on the exceptional affinity of streptavidin for biotin to very efficiently isolate them. While BioID performs better than AP for identifying transient or weak interactions, both AP- and BioID-mass spectrometry approaches provide an overview of all possible interactions a given protein may have. However, they do not provide information on the context of each identified PPI. Indeed, most proteins are typically part of several complexes, corresponding to distinct maturation steps or different functional units. To address this common limitation of both methods, we have engineered a protein-fragments complementation assay based on the BirA* enzyme. In this assay, two inactive fragments of BirA* can reassemble into an active enzyme when brought in close proximity by two interacting proteins to which they are fused. The resulting split-BioID assay thus allows the labeling of proteins that assemble around a pair of interacting proteins. Provided these two only interact in a given context, split-BioID then allows the analysis of specific context-dependent functional units in their native cellular environment. Here, we provide a step-by-step protocol to test and apply split-BioID to a pair of interacting proteins.

We provide a step-by-step protocol for split-BioID, a protein fragments-complementation assay based on the proximity-labeling technique BioID. Activated on the interaction of two given proteins, it allows the proteomics analysis of context-dependent protein complexes in their native cellular environment. The method is simple, cost-effective and only requires standard laboratory equipment.

BioID 细胞环境中特异蛋白复合物的分离-蛋白质组学分析

To complement existing affinity purification (AP) approaches for the identification of protein-protein interactions (PPI), enzymes have been introduced ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

High-resolution Spatiotemporal Analysis of Receptor Dynamics by Single-molecule Fluorescence Microscopy

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., kinetics, coexistence of different states and populations, transient interactions), which are typically hidden in ensemble measurements, such as those obtained with standard biochemical or microscopy methods. Thus, dynamic events, such as receptor-receptor interactions, can be followed in real time in a living cell with high spatiotemporal resolution. This protocol describes a method based on labeling with small and bright organic fluorophores and total internal reflection fluorescence (TIRF) microscopy to directly visualize single receptors on the surface of living cells. This approach allows one to precisely localize receptors, measure the size of receptor complexes, and capture dynamic events such as transient receptor-receptor interactions. The protocol provides a detailed description of how to perform a single-molecule experiment, including sample preparation, image acquisition and image analysis. As an example, the application of this method to analyze two G-protein-coupled receptors, i.e., &#946;2-adrenergic and &#947;-aminobutyric acid type B (GABAB) receptor, is reported. The protocol can be adapted to other membrane proteins and different cell models, transfection methods and labeling strategies.

This protocol describes how to use total internal reflection fluorescence microscopy to visualize and track single receptors on the surface of living cells and thereby analyze receptor lateral mobility, size of receptor complexes as well as to visualize transient receptor-receptor interactions. This protocol can be extended to other membrane proteins.

受体动力学通过单分子荧光显微镜的高分辨率时空分析

Single-molecule microscopy is emerging as a powerful approach to analyze the behavior of signaling molecules, in particular concerning those aspect (e.g., ...

生物工程

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

可视化的活细菌细胞蛋白-DNA相互作用使用光活化的单分子跟踪

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

免疫与感染

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

光敏定位显微镜与双分子荧光互补（附设-PALM）

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen Presenting Cells (APCs). This is despite T-cell antigen receptors (TCRs) exerting usually a moderate affinity (&#181;M range) to antigen when binding is measured in vitro1. In view of the molecular and cellular parameters contributing to T-cell antigen sensitivity, a microscopy-based methodology has been developed as a means to monitor TCR-pMHC binding in situ, as it occurs within the synapse of a live T-cell and an artificial and functionalized glass-supported planar lipid bilayer (SLB), which mimics the cell membrane of an Antigen presenting Cell (APC) 2. Measurements are based on F&#246;rster Resonance Energy Transfer (FRET) between a blue- and red-shifted fluorescent dye attached to the TCR and the pMHC. Because the efficiency of FRET is inversely proportional to the sixth power of the inter-dye distance, one can employ FRET signals to visualize synaptic TCR-pMHC binding. The sensitive of the microscopy approach supports detection of single molecule FRET events. This allows to determine the affinity and off-rate of synaptic TCR-pMHC interactions and in turn to interpolate the on-rate of binding. Analogous assays could be applied to measure other receptor-ligand interactions in their native environment.

Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay

This manuscript describes how to conduct (single molecule) F&#246;rster Resonance Energy Transfer (FRET)- based assays to measure the binding dynamics between T-cell antigen receptor (TCR) and antigenic peptide-loaded MHC molecules as they occur within the immunological synapse of a T-cell in contact with a functionalized planar supported lipid bilayer.

测量TCR-的pMHC结合<em&gt;原位</em&gt;使用基于FRET的显微镜分析

T-cells are remarkably specific and effective when recognizing antigens in the form of peptides embedded in MHC molecules (pMHC) on the surface of Antigen ...

Fluorescent Labeling of COS-7 Expressing SNAP-tag Fusion Proteins for Live Cell Imaging

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. These systems offer a broad selection of fluorescent substrates optimized for a range of imaging instrumentation. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again. There are two steps to using this system: cloning and expression of the protein of interest as a SNAP-tag fusion, and labeling of the fusion with the SNAP-tag substrate of choice. The SNAP-tag is a small protein based on human O6-alkylguanine-DNA-alkyltransferase (hAGT), a DNA repair protein. SNAP-tag labels are dyes conjugated to guanine or chloropyrimidine leaving groups via a benzyl linker. In the labeling reaction, the substituted benzyl group of the substrate is covalently attached to the SNAP-tag. CLIP-tag is a modified version of SNAP-tag, engineered to react with benzylcytosine rather than benzylguanine derivatives. When used in conjunction with SNAP-tag, CLIP-tag enables the orthogonal and complementary labeling of two proteins simultaneously in the same cells.

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest in live cells. Once cloned and expressed, the tagged protein can be used with a variety of substrates for numerous downstream applications without having to clone again.

对活细胞成像的SNAP - Tag融合蛋白的荧光标记的COS - 7

SNAP-tag and CLIP-tag protein labeling systems enable the specific, covalent attachment of molecules, including fluorescent dyes, to a protein of interest ...

生物学

Multi-color Localization Microscopy of Single Membrane Proteins in Organelles of Live Mammalian Cells

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a super-resolution technique that allows for the determination of the microcompartments that are accessible for proteins by generating localization and tracking maps of these proteins. Moreover, by multi-color localization microscopy, the localization and tracking profiles of proteins in different subcompartments are obtained simultaneously. The technique is specific for live cells and is based on the repetitive imaging of single mobile membrane proteins. Proteins of interest are genetically fused with specific, so-called self-labeling tags. These tags are enzymes that react with a substrate in a covalent manner. Conjugated to these substrates are fluorescent dyes. Reaction of the enzyme-tagged proteins with the fluorescence labeled substrates results in labeled proteins. Here, Tetramethylrhodamine (TMR) and Silicon Rhodamine (SiR) are used as fluorescent dyes attached to the substrates of the enzymes. By using substrate concentrations in the pM to nM range, sub-stoichiometric labeling is achieved that results in distinct signals. These signals are localized with ~15&#8211;27 nm precision. The technique allows for multi-color imaging of single molecules, whereby the number of colors is limited by the available membrane-permeable dyes and the repertoire of self-labeling enzymes. We show the feasibility of the technique by determining the localization of the quality control enzyme (Pten)-induced kinase 1 (PINK1) in different mitochondrial compartments during its processing in relation to other membrane proteins. The test for true physical interactions between differently labeled single proteins by single molecule FRET or co-tracking is restricted, though, because the low labeling degrees decrease the probability for having two adjacent proteins labeled at the same time. While the technique is strong for imaging proteins in membrane compartments, in most cases it is not appropriate to determine the localization of highly mobile soluble proteins.

Here, we present a protocol for multi-color localization of single membrane proteins in organelles of live cells. To attach fluorophores, self-labeling proteins are used. Proteins, located in different membranes compartments of the same organelle, can be localized with a precision of ~18 nm.

活体哺乳动物细胞器中单膜蛋白的多色定位显微术

Knowledge about the localization of proteins in cellular subcompartments is crucial to understand their specific function. Here, we present a ...

基于活细胞单分子成像的蛋白质相互作用定量

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number of cellular functions. Characterizing LLPS in live cells is also important because aberrant condensation has been linked to numerous diseases, including cancers and neurodegenerative disorders. LLPS is often driven by selective, transient, and multivalent interactions between intrinsically disordered proteins. Of great interest are the interaction dynamics of proteins participating in LLPS, which are well-summarized by measurements of their binding residence time (RT), that is, the amount of time they spend bound within condensates. Here, we present a method based on live-cell single-molecule imaging that allows us to measure the mean RT of a specific protein within condensates. We simultaneously visualize individual protein molecules and the condensates with which they associate, use single-particle tracking (SPT) to plot single-molecule trajectories, and then fit the trajectories to a model of protein-droplet binding to extract the mean RT of the protein. Finally, we show representative results where this single-molecule imaging method was applied to compare the mean RTs of a protein at its LLPS condensates when fused and unfused to an oligomerizing domain. This protocol is broadly applicable to measuring the interaction dynamics of any protein that participates in LLPS.

作者聚焦:活人细胞中蛋白质-凝聚物动力学的评估

Many intrinsically disordered proteins have been shown to participate in the formation of highly dynamic biomolecular condensates, a behavior important for numerous cellular processes. Here, we present a single-molecule imaging-based method for quantifying the dynamics by which proteins interact with each other in biomolecular condensates in live cells.

生物分子缩合物中蛋白质相互作用动力学的单分子测量

Biomolecular condensates formed via liquid-liquid phase separation (LLPS) have been considered critical in cellular organization and an increasing number ...