Name: optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells
Uploaded: 2018-04-09T14:30:00
Description: Watch this Scientific Journal Video about Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells at JoVE.

这项工作是由德意志 Forschungsgemeinschaft (DFG) 根据赠款 CO822/2-1 (艾美诺特计划) 和 CO822/3-1 到中

1. 基于荧光的筛选效率 (图 1)
<ol><li>维持 Dulbecco 修饰鹰培养基中的 HEK293 细胞 (DMEM; 高葡萄糖, 4 毫米谷氨酰胺, 丙酮酸) 补充 10% (v/v) 胎牛血清 (血清), 100 U/毫升青霉素和100µg/毫升链霉素在37°c, 95% 湿度和 5% CO2。</li>
	<li>在转染前的前一天将细胞播种。<ol>
<li>分离细胞为5分钟在37°c 在0.05% 胰蛋白酶或 PBS 补充与0.5 毫米 EDTA。使用1毫升胰蛋白酶/EDTA 的10厘米菜。用10卷的...

该协议描述了一个简单而可靠的方法, 以评估正交对的效率, 将 ncAAs 纳入哺乳动物细胞表达的蛋白质。这种方法的主要优点是, 在广泛使用的基于资产管制系统的测试中, 它允许同时准备和测量大量的样本, 并提供了使用普通软件可以轻松分析的数据。在哺乳动物细胞中进行正交对分析的中等吞吐量方法的有效性对于新的正交对的发展和现有的改进是非常重要的。事实上, 不可能通过生成大型随机库...

化学探针在蛋白质中的遗传结合是一种有效的方法, 可以直接在活细胞的本机语境中研究蛋白质功能的结构和动态方面。如今, 数以百计的非规范氨基酸 (ncAAs) 配备了最不同的化学基团, 可以通过生物合成1,2,3,4,专门纳入蛋白质。在他们之间, 你发现光敏 ncAAs 例如相片交联剂 5, 相片笼6, 7, 8, 9 和相片转换氨基酸10,11, 含氨基酸的烯烃和炔烃无催化剂 bioorthogonal 化学2,12,13,14,15,16 、17、携带丹磺酰18、香豆素9、19、prodan20、21显影的氨基酸以及配备其他生物物理探针的氨基酸作为以及与后平移修改1,2,3,4,22,23,24,25。
ncaa 的遗传编码是由一个专门的氨基酰 tRNA 合成酶 (AARS) 配对的同源抑制-tRNA, 其中结合了 ncaa 的反应琥珀停止密码在定期核糖体合成。ncAARS/tRNA 对被设计成在宿主有机体中是正交的,即不与内生对进行交叉交谈。该技术在原核和真核宿主中都有很好的建立, 并且很容易适用于哺乳动物细胞。对 ncAA 纳入哺乳动物细胞是基于三主要正交系统: tyrosyl 系统, 结合 TyrRS 从大肠杆菌26与 tyrosyl 琥珀色抑制器从 B. 嗜热脂肪 27(EcTyrRS/Bst山药对),大肠杆菌leucyl 系统 (EcLeuRS/tRNA列在CUA对)6,18,28和古细菌 pyrrolysyl 系统 (PylRS/tRNAPyl对)3, 藉以 tRNAPyl是自然琥珀抑。一般来说, 每个 ncAA 都被一个专业的 ncAARS 认可。根据 ncaa 的结构, ncAARS 是通过 TyrRS、LeuRS 或 PylRS 的定向进化获得的, 尽管一些合成酶系可以接受不止一个 ncaa。
将正交对通过简单地使用质粒载体导入细胞中。最常见和有效的质粒是 bicistronic 和编码的合成酶和 tRNA 形成正交对29。第二种质粒编码的兴趣的蛋白质, 轴承一个琥珀密码子在指定的修改地点被联合转染。ncAA 只是添加到细胞生长培养基中。然而, 不同的专业群体经常使用不同的质粒结构, 即使是同一个 ncAA 的合并。构造在基因的排列不同在载体, 合成酶的类型, 密码子使用在合成酶基因, 促进者用法, tRNA 的变异和 tRNA 表达盒的数量。另外, 由于不同合成酶系的催化效率、tRNA 的质量和其他因素的不同, 不同 ncAAs 的结合效率可能会有很大的差异,如30。因此, 在手边有一个快速而可靠的方法来评估正交对的效率, 无论是选择最适合的系统为理想的应用和执行一些优化步骤, 以提高整体蛋白表达收益 率。
我们已经建立了一个简单和稳健的荧光检测方法来评估正交对29 (图 1) 的效率。在该检测中, 细胞与质粒编码的正交对, 连同 bicistronic 报告质粒编码两个绿色荧光蛋白轴承一个琥珀停止密码子在允许的位置 (EGFP标记) 和mCherry 基因。全细胞裂解物的红色和绿色荧光在一个96井板上的平板阅读器上以不同的通道读出。绿色荧光的强度与琥珀色抑制的效率直接相关, 而红荧光强度则直接估计出所测样品的大小和转染效率。关于基于荧光辅助细胞分类的类似化验, 请阅读31、32, 该化验结果立即全面评估了整个细胞的蛋白质表达, 这更代表通常的实验条件, 并提供一个更容易的数据获取和处理标准软件。总的来说, 该方法的主要优点是可以同时分析大量样品中的介质。利用这一方法, 我们筛选了一个合理设计的抑制 tRNAs 库, 以提高 Pyl 正交系统30的效率。这项工作描述的实验协议, 以执行这一试验, 并显示其应用的例子, 包括优化的正交对组合的照片交联的叠氮基苯丙氨酸 (Azi) 和比较采用不同氨基酸的效率 (图 2)。
在过去的几年中, ncAA 的工具已经被证明是非常强大的研究 G 蛋白耦合受体的结构和功能方面 (受体)33,34,35,36,37,38. 在人类中, 受体形成了一个大家庭的膜受体 (800 名成员), 并代表治疗药物的主要目标。受体的直接结构表征仍然具有挑战性, 对其进行研究需要补充生物化学方法。我们率先使用的照片交联 ncAAs 地图 GPCR 表面和发现配体绑定口袋34。利用我们的优化系统 Azi 合并, 我们系统地纳入 Azi 整个 juxtamembrane 领域的 GPCR 直接在活体哺乳动物细胞。在紫外线照射下, Azi 形成一种高度反应的 nitrene 物种, 共价键捕获相邻分子。当配体添加到系统中时, Azi 作为近距离探针来揭示受体的位置接近束缚配体。这样, 在 B 类 GPCR 内促肾上腺皮质激素释放因子受体类型 1 (CRF1R) 33 上首次公布了神经肽激素 Urocortin I (Ucn1) 的结合模式。最近, 我们在同一受体38上公开了不同的激动剂和拮抗器的结合模式。其他受体39、40、41、42的其他肽和小分子配体的 orthosteric 和构结合点也采用了类似的方法。本文介绍了在 GPCR 表面光交联测绘实验室中应用的实验协议。该方法相对快速, 简单, 不需要任何特殊设备, 因此适用于标准生物化学实验室。重要的是, 该方法不仅提供了一个有价值的工具, 不仅可以识别3D 结构数据稀缺的配体结合点, 而且还可以补充现有的体外数据, 并从完全后 translationally 修改后的受体中得到信息。活细胞的生理环境。
新近开发的新型 ncAAs 轴承, 适用于超快无催化剂 bioorthogonal 化学的侧链化学基团, 已开辟了将最后一代显影安装到蛋白质中的超级分辨率成像的可能性。直接在活单元格2,43上。 此类化学锚包括 SCOK14中的紧张 cyclooctyne, 双环 [6.1. 0] 壬炔BCNK12, 17, 和跨 cyclooctenes 在 TCO *K13, 15, 17 等 ncAAs窝藏冰片16,17,44或 cyclopropene45,46基团。bioorthogonal 化学的笨重 ncAAs 由 PylRS 的变体组成, 通常表示为 PylRSAF (指示m Y271A Y349F 中的突变 barkeri 和 PylRS), 以及其他即席进化 ncAARSs 17, 44. bioorthogonal 锚与四嗪类试剂47 通过逆电子需求翅果-桤木 cycloaddition 的反应, 在几分钟43, 48 内给出高标号率。然而, 这一强大的方法, 以标签受体是一个挑战, 由于整体效率低的正交 ncAA 公司合并系统。利用我们的增强 Pyl 系统, 我们最近展示了这种氨基酸的高产量纳入受体和超快 GPCR 标记在活体哺乳动物细胞表面30。标记的受体仍然功能, 因为他们的生理内化后激活受体与激动剂。本文介绍了将 bioorthogonal 锚定为受体的实验协议, 以及下面的标记步骤。用小亮显影装备受体是通过先进显微技术研究活细胞 GPCR 结构动力学的第一个基本步骤。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acryamide/Bisacrylamide 30% (37,5:1)</td>
			<td>Carl Roth</td>
			<td>3029.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Carl Roth</td>
			<td>9592.2</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Azidophenylalanine (Azi)</td>
			<td>Bachem</td>
			<td>F-3075.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma Aldrich</td>
			<td>B6768</td>
			<td></td>
		</tr>
		<tr>
			<td>Bromphenolblue</td>
			<td>Sigma-Aldrich</td>
			<td>B0126-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumine (BSA)</td>
			<td>Carl Roth</td>
			<td>8076.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbobenzyloxy-L-lysine (Lys(Z))</td>
			<td>NovaBiochem</td>
			<td>8540430100</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclooctyne-L-lysine (SCOK)</td>
			<td>Sichem</td>
			<td>SC-8000</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>41966052</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Carl Roth</td>
			<td>A994.2</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Carl Roth</td>
			<td>6908.1</td>
			<td></td>
		</tr>
		<tr>
			<td>enhanced chemiluminescence reagent (ECL)&#160;</td>
			<td>home-made</td>
			<td></td>
			<td>10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l&#160; p-coumaric acid in DMSO ; 30&#160;% H2O2 (1,000 : 100 : 0.3) [Quelle Laborjounal]</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Carl Roth</td>
			<td>8043.1</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Carl Roth</td>
			<td>3054.1</td>
			<td></td>
		</tr>
		<tr>
			<td>endo-bicyclo[6.1.0]nonyne-L-lysine (BCNK)</td>
			<td>Sichem</td>
			<td>SC-8014</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>10270106</td>
			<td></td>
		</tr>
		<tr>
			<td>FluoroBrite DMEM</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>A1896701</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Carl Roth</td>
			<td>7533.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycin</td>
			<td>Carl Roth</td>
			<td>3908.3</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Carl Roth</td>
			<td>9105.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Sigma Aldrich</td>
			<td>B2261</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Carl Roth</td>
			<td>6781.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000&#160;</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Luminol</td>
			<td>Applichem</td>
			<td>A2185,0005</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Carl Roth</td>
			<td>0082.3</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Carl Roth</td>
			<td>2189.2</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Carl Roth</td>
			<td>HN00.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Na-Lactate</td>
			<td>Sigma-Aldrich</td>
			<td>71718-10G</td>
			<td></td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Gr&#252;ssing</td>
			<td>121551000</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>P5493-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>p-Coumaric acid</td>
			<td>Sigma-Aldrich</td>
			<td>C9008-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>poly-D-lysine hydrobromide</td>
			<td>Corning</td>
			<td>354210</td>
			<td></td>
		</tr>
		<tr>
			<td>PEI</td>
			<td>Polysciences</td>
			<td>23966</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>11548876 (15140-122)</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Carl Roth</td>
			<td>6367.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PNGase F</td>
			<td>NEB</td>
			<td>P0704L</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF membrane Immobilon-P</td>
			<td>Millipore</td>
			<td>IPVH00010</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk Powder&#160;</td>
			<td>Sigma</td>
			<td>70166</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Carl Roth</td>
			<td>CN30.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrazine-Cy3</td>
			<td>Jena Bioscience</td>
			<td>CLK-014-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Carl Roth</td>
			<td>2367.3</td>
			<td></td>
		</tr>
		<tr>
			<td>trans-Cyclooctene-L-lysine (TCO*K)</td>
			<td>Sichem</td>
			<td>SC-8008</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Carl Roth</td>
			<td>3051.4</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin 2.5%</td>
			<td>Thermo Fisher (Gibco)</td>
			<td>15090046</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Carl Roth</td>
			<td>9127.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Wasserstoffperoxid (30%)</td>
			<td>Merck</td>
			<td>1.07210.0250</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell lines</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-305</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>German Collection of Microorganisms and Cell Cultures GmbH (DSMZ)</td>
			<td>ACC-635</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Crosslinker Bio-Link 365 nm</td>
			<td>Bio-Budget Technologies GmbH</td>
			<td>40-BLX-E365</td>
			<td>5 x 8 Watt tubes</td>
		</tr>
		<tr>
			<td>Plate Reader BMG LABTECH FLUOstar Omega&#160;</td>
			<td>BMG LABTECH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmids</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid E2AziRS</td>
			<td>The huminized gene for E2AziRS was synthesized by Geneart (Life Technologies)</td>
			<td></td>
			<td>Plasmid containing 4 tandem copies of the suppressor tRNA Bst-Yam driven by the human U6 promoter and one copy of a humanized gene for the enhanced variant of the Azi-tRNA synthetase (EAziRS) driven by a PGK promoter</td>
		</tr>
		<tr>
			<td>POI-TAG mutant plasmids</td>
			<td></td>
			<td></td>
			<td>Plasmid encoding the POI driven by the CMV promoter, C-terminally fused to the FLAG-tag, bearing a TAG codon at the desired position&#160;</td>
		</tr>
		<tr>
			<td>CRF1R-95TAG-EGFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>HA-PTH1R-79TAG-CFP</td>
			<td></td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Arrestin3-FLAG</td>
			<td>Synthesized by Genart (Life Technologies)</td>
			<td></td>
			<td>Cloned in the MCS of pcDNA3.1</td>
		</tr>
		<tr>
			<td>Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-FLAG-HRP M2 antibody conjugate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>A8592&#160;</td>
			<td>monoclonal, produced in mouse clone M2</td>
		</tr>
		<tr>
			<td>Goat-anti-rabbit-HRP antibody</td>
			<td>Santa Cruz</td>
			<td>sc-2004</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit-anti-CRF antibody</td>
			<td>home-made</td>
			<td>PBL #rC69</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
		<tr>
			<td>Rabbit-anti-Ucn1 antibody</td>
			<td>home-made</td>
			<td>PBL #5779</td>
			<td>polyclonal [Turnbull]</td>
		</tr>
	</tbody>
</table>

optimizing the genetic incorporation of chemical probes into gpcrs for photo-crosslinking mapping and bioorthogonal chemistry in live mammalian cells

非规范氨基酸 (ncAAs) 的基因融合通过琥珀停止密码子抑制是一个强大的技术, 安装人工探针和反应基团直接在蛋白质的活细胞。每个 ncAA 都由一个专用的正交抑制-tRNA/氨基酰 tRNA 合成酶 (AARS) 对导入宿主有机体。不同 ncAAs 的并网效率有很大差异, 在某些情况下是不理想的。通过操作 AARS 或 tRNA 可以提高正交对。然而, tRNA 或 AARS 的定向进化使用大型图书馆和死/活选择方法是不可行的哺乳动物细胞。在此基础上, 提出了一种简便、稳健的荧光检测方法, 用于评价哺乳动物细胞中正交对的有效性。该化验允许在合理的时间内进行适量的 AARS/tRNA 变种的筛查。利用这一方法生成新的 tRNAs, 显著提高 pyrrolysine 正交系统的效率, 以及 ncAAs 在 G 蛋白耦合受体 (受体) 的研究中的应用, 这是 ncAA 的挑战对象。诱变。首先, 系统地将一张照片交联的 ncAA 在一个受体的胞外表面, 在完整的受体的结合部位直接映射到活细胞中。其次, 通过将最后一代 ncAAs 加入 GPCR, 证明了用荧光染料进行超快无催化剂受体标记, 利用 bioorthogonal 应变促进逆翅果桤木 cycloaddition (SPIEDAC) 在活细胞上的作用。由于 ncAAs 可以被广泛地应用于任何蛋白质, 它的大小, 该方法是普遍感兴趣的一些应用。此外, ncAA 公司不需要任何特殊的设备, 很容易在标准生物化学实验室进行。

荧光检测的轮廓在图 1中描述。这项化验在三宗申请中使用。首先, 对 Pyl 正交对中的赖氨酸 (中行) 的一些 tRNA 变体进行筛选。赖氨酸 (中行) 是一种与 Pyl 类似的氨基酸 sterically。由于 Pyl 在商业上不可用, 赖氨酸 (中行) 通常被用作 PylRS 的标准基板。筛选的 tRNAs 基于 tRNAPyl。每个 tRNA 变种在回路和茎的单基或基对的突变承担合理设计, 以提高 tR...

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

本文提出了一种简便的荧光分析方法, 用于评价将非规范氨基酸 (ncAAs) 纳入哺乳动物细胞表达的蛋白质中的氨基酰 tRNA 合成酶/tRNA 对的效率。介绍了 ncAAs 在 G 蛋白耦合受体 (受体) 研究中的应用, 包括结合位点的光交联映射和活细胞的 bioorthogonal GPCR 标记。

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 ...

The overall goal of this protocol is to enhance the incorporation rate of non-canonical amino acids in mammalian cells to tackle biological questions in live cell settings including mapping of protein-protein interaction surfaces, and bioorthogonal labeling of surface receptors. Using amber codon suppression, different non-canonical amino acids are incorporated with different efficiencies. We established fluorescence assay to easily compare non-canonical amino acids incorporation rates in mammalian cells within a short time.The systematic incorporation of photo-crosslinking, non-canonical amino acids through our GPCR surfaces allows mapping of ligand binding sites and provides precious information of the intake receptor directly from the life cell. By incorporating last generation, non-canonical amino acids for bioorthogonal chemistry, we achieve site-specific, super fast, catalyst free labeling of GPCRs with small, bright fluorophores for high-end imaging applications in live mammalian cells. First, seed 600, 000 HEK 293 cells in two milliliters of complete growth medium per well in two six-well plates.Prepare as many wells as the number of samples and two additional wells for the wild type EGFP and a mock transfected sample respectively. One hour prior to transfection, add the appropriate amount of freshly prepared non-canonical amino acid stock solution to all wells for a final amino acid concentration of 0.25 to 0.5 millimolar. To transfect the cells, mix one microgram of plasmid DNA and coding for the synthetase t-RNA pair to be tested with one microgram of reporter plasmid DNA in a microcentrifuge tube.In separate tubes, prepare an identical transfection using the EGFP wild type reference and a mock transfection. Next, add 100 microliters of lactate buffered saline to each tube containing the DNA. After briefly mixing, add six microliters of one microgram per microliter PEI and lactate buffered saline to each tube.Vortex immediately, and then incubate at room temperature for 10 to 15 minutes. Transfer 400 microliters of cell medium from each well to the DNA PEI mixture to neutralize the pH. Then, dribble the DNA mixture onto the cells.Harvest the cells 48 hours post-transfection by aspirating the medium and rinsing the cells once with two milliliters of pre-warmed PBS. Add 800 microliters of PBS supplemented with 0.5 millimolar EDTA, and incubate for 20 minutes at 37 degrees Celsius. After incubation, detach and suspend the cells by pipetting up and down.Next, transfer the cell suspension into microcentrifuge tubes containing 200 microliters of PBS supplemented with five millimolar magnesium chloride. Centrifuge the samples for two minutes at 800 times gravity. When finished, discard the supernatant.Now, add 100 microliters of Tris lysis buffer to the cell pellets and incubate on ice for 30 minutes. To facilitate lysis, vortex every five minutes. Spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity.Transfer 90 microliters of the supernatant into black 96-well plates. Then, measure EGFP and mCherry fluorescence using a plate reader equipped with the fluorescence module. Seed 500, 000 293 t-cells in two milliliters of complete growth medium per well in two six-well plates.For each position to be screened, prepare one well per ligand, plus one well for the binding control. One hour prior to transfection, add the photo-crosslinker, p-azidophenylalanine, or Azi, to all wells to a final concentration of 0.5 millimolar. Transfect a total amount of two micrograms of DNA per well using one microgram of plasmid and coding for the flag tagged GPCR bearing a tag codon at the desired position, and one microgram of the plasmid and coding for the orthogonal pair dedicated to Azi.At 48 hours post-transfection, prepare a 1, 000x ligand stock solution by dissolving the peptide ligand at a concentration of 100 micromolar in DMSO. Dilute the ligand stock solution one to 1, 000 in binding buffer. Replace the cell medium with one milliliter of the ligand solution and incubate the samples for 10 minutes at room temperature.Following incubation, irradiate the cells for 20 minutes in a UV cross-linker at 365 nanometers and five centimeter distance to the cells. When finished, detach the cells by pipetting and transfer them into a microcentrifuge tube. Pellet the cells for three minutes at 800 times gravity.After discarding the supernatant, resuspend the cell pellets in 50 microliters of HEPES dissociation buffer, or HDB, containing a cocktail of protease inhibitors. Then, flash freeze the cells in liquid nitrogen. Next, thaw the cells in a water bath at 37 degrees Celsius for 30 to 45 seconds, and then vortex briefly.Pellet the membranes for 10 minutes at four degrees Celsius and 2500 times gravity. When finished, discard the supernatant which contains the bulk of cytosolic proteins. Thoroughly resuspend the pellets in 50 microliters of HEPES lysis buffer using a pipet.Then, lyse the cells for 30 minutes on ice, vortexing every five minutes. Following this, spin down the cell debris for 10 minutes at four degrees Celsius and 14, 000 times gravity. Immediately transfer each supernatant to a fresh reaction tube for western blot analysis.When the GPCR is glycosylated and faint or smeared bands are a problem in western blot analysis, deglycosylate the samples with PNGase F to increase signal intensity and sharpen the bands. Then, resolve the samples on standard SDS page and blot transfer proteins to a PVDF membrane. After blocking the membrane, probe it with an anti-ligand antibody to detect the occurrence of cross-linking.To detect the expression level of the Azi-GPCR, probe the membrane with a commercial anti-flag antibody. After washing the membrane, soak it in enhanced chemiluminescence solution, and visualize the signals in the dark. Seed 100, 000 HEK 293 t-cells in 600 microliters of dye-free, complete DMEM per well of a microscopy slide equipped with four wells.One hour prior to transfection, prepare a fresh 100 millimolar stock solution of TCOK and 0.2 molar sodium hydroxide and 15%DMSO. Mix three microliters of the TCOK stock solution with 12 microliters of one molar, pH 7.4 HEPES buffer. Gently add the solution to each well for a final TCOK concentration of 0.5 millimolar.In a microcentrifuge tube, dilute 200 nanograms of the plasmid and coding for the receptor containing the amber codon and 200 nanograms of the plasmid and coding for the orthogonal pair in 50 microliters of medium. Dilute 1.25 microliters of lipofectamine 2, 000 in 50 microliters of dye-free, serum-free, and antibiotic-free medium, and add the solution to the DNA solution. Vortex immediately and incubate for five to 10 minutes at room temperature.Then, add the DNA lipid complexes to the cells. At 24 hours post-transfection, transfer 100 microliters of medium from each well into a 1.5 milliliter reaction tube. Add 1.8 microliters of an orange fluorescent dye tetrazine stock solution, and 0.3 microliters of a DNA staining dye stock solution.Transfer the medium containing the dyes back to the well and incubate for five minutes at 37 degrees Celsius. Aspirate the medium, and gently rinse the cells twice with PBS to remove the excess dye. Then, add 600 microliters of pre-heated, complete dye-free growth medium to the cells.Now, visualize the labeled receptors under 63x magnification using filters appropriate for GFP orange fluorescent dye and DNA-staining dye. After adding peptide agonist stock solution to the cells, observe the internalization under the microscope using the aforementioned filters and take pictures after the clearly detectable occurrence of internalization. Different tRNA's give different suppression efficiency, which is clearly reflected in the amount of measured fluorescence.Azi incorporation into CRF1R was highly enhanced when using the codon-optimized Azi-tRNA synthetase gene compared to the native gene, showing that even a moderate improvement for a soluble protein can have a greater impact on the expression level of more challenging membrane proteins. The optimized system for Azi incorporation, including the humanized E2 Azi RS gene was deployed to map the binding pocket of the CRF1R and to find binding paths of five ligands. A band at the correct size of the crosslinking product in the western blot reveals that the position lies in the proximity of the ligand within the ligand receptor complex.Multiple crosslinking hits with all ligands tested revealed distinct binding patterns for the peptide Agonists and Antagonists. Both fluorescence and western blot data suggests that TCOK is the non-canonical amino acids for bioorthogonal chemistry that gets incorporated with the highest efficiency of the AF variant of the paralysine tRNA synthetase. TCOK was incorporated into a CRF1R EGFP fusion protein and enabled installing a small, bright fluorophore on the receptor.After adding a peptide Agaonist, fluorescent compartments were observed throughout the cytosol, revealing the physiological process of GPCR internalization. To do a fluorescence assay, we presented here is a simple and effective tool to evaluate and optimize the efficiency of a synthetase TI and API in mammalian cells. High incorporation rates are crucial for applying non-canonical amino acids to investigate challenging biological questions.Highly efficient incorporation of crosslinking non-canonical amino acids enables extensive surface mapping of virtually any protein, including membrane receptors or low-banded proteins. The method reveals the topology of interaction interfaces which can be further analyzed to obtain accurate molecular models. High incorporation efficiency of last generation, non-canonical amino acids for bioorthogonal chemistry allows us to equip GPCRs with small, bright fluorophores, thus leading the way towards the study of GPCRs structural dynamics in live cells via advanced microscopy techniques.

Research

JoVE Journal

Chemistry

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

隔离和脂质A的化学表征革兰氏阴性菌

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS ...

科研

化学

A Simple and Rapid Protocol for Measuring Neutral Lipids in Algal Cells Using Fluorescence

一种简单快速的协议用荧光测量中性脂肪在藻细胞

Algae are considered excellent candidates for renewable fuel sources due to their natural lipid storage capabilities. Robust monitoring of algal ...

Preparing Adherent Cells for X-ray Fluorescence Imaging by Chemical Fixation

化学固定准备贴壁细胞的X射线荧光成像

X-ray fluorescence imaging allows us to non-destructively measure the spatial distribution and concentration of multiple elements simultaneously over ...

Chemical Vapor Deposition of an Organic Magnet, Vanadium Tetracyanoethylene

化学气相沉积的有机磁体，钒四氰

Recent progress in the field of organic materials has yielded devices such as organic light emitting diodes (OLEDs) which have advantages not found in ...

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical (Methylation) and Physical (Mass Spectrometry, Nuclear Magnetic Resonance) Techniques

Sequencing of Plant Wall Heteroxylans Using Enzymic, Chemical Methylation and Physical Mass Spectrometry, Nuclear Magnetic Resonance Techniques

使用酶法，化学（甲基化）和物理（质谱，核磁共振）技术，植物墙Heteroxylans测序

This protocol describes the specific techniques used for the characterization of reducing end (RE) and internal region glycosyl sequence(s) of ...

Immobilization of Multi-biocatalysts in Alginate Beads for Cofactor Regeneration and Improved Reusability

藻酸盐珠多生物催化剂辅因子再生和可重用性改进的固定化

We have recently developed a simple, reusable and coupled whole-cell biocatalytic system with the capability of cofactor regeneration and biocatalyst ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

蛋白质生物缀合物的合成<em&gt;通过</em&gt;半胱氨酸马来酰亚胺化学

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

The Preparation and Properties of Thermo-reversibly Cross-linked Rubber Via Diels-Alder Chemistry

热可逆交联橡胶通过的Diels-Alder反应的化学制备与性能

A method for using Diels Alder thermo-reversible chemistry as cross-linking tool for rubber products is demonstrated. In this work, a commercial ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

洞察氨基酸和多肽与无机材料的使用相互作用的单分子力谱

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

Preparation and Evaluation of 99mTc-labeled Tridentate Chelates for Pre-targeting Using Bioorthogonal Chemistry

制备和评价<sup&gt;99米</sup&gt;锝标记的三齿螯合物预靶向使用生物正交化学

Pre-targeting combined with bioorthogonal chemistry is emerging as an effective way to create new radiopharmaceuticals. Of the methods available, the ...