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卷的完全培养基淬火, 并通过吹打并用重悬细胞。使用 hemocytometer49计算挂起的单元格数。</li>
		<li>种子 6.0 x 105 HEK293 细胞每井6井板在2毫升完全生长培养基。分别为野生型 EGFP 和模拟转染样品准备尽可能多的井作为样品数量和另外两个井。</li>
	</ol>
</li>
	<li>显微镜下控制汇流 (由细胞占据的区域)。染细胞在70% 汇合使用聚乙烯亚胺 (裴) 试剂。<ol>
<li>1h. 在转染之前, 加入适当数量的新的 ncaa 股票解决方案, 所有的水井为最终的 ncaa 浓度为 0.25-0. 5 毫米。将 ncaa 纳入所有水井, 包括野生型阳性对照和模拟转染细胞, 以防止由 ncaa 对细胞生长的影响可能引起的荧光信号的差异。 注意:要准备库存解决方案, 用 0.2-0.5 米氢氧化钠将 ncAA 分解为 0.1-0.5 米。然而, 在使用前, 一些 ncAAs 可能需要在亚砜和/或中和四卷1米 HEPES (pH 7.4) 的初始增溶。通常, 制造商建议制定一套库存解决方案的协议。</li>
		<li>在离心管中, 混合1µg 质粒 dna 编码的 ncAARS/tRNA 对测试与1µg 的报告质粒 dna (pcDNA3.0 EGFP183TAG-mCherry)。在不同的管, 准备一个相同的转染使用 EGFP 野生型参考和模拟转染。 注意:ncAARS/tRNA 对的质粒编码中嵌入的 tRNA 盒的拷贝数取决于应用。为便于克隆, 在筛选不同的 tRNAs 时建议使用 1 tRNA 拷贝, 而在测试不同的 ncAARS 或将不同的 ncAAs 由同一正交对组合在一起时, 建议使用4个拷贝 (尽管不是绝对必要)。</li>
		<li>每个包含 DNA 的管中添加100µL 乳酸缓冲盐水 (磅), 含20毫米乳酸钠在 pH 4.0 和150毫米氯化钠。简单地混合。</li>
		<li>每管包含的 DNA 在 lbs 添加6µL 1 µg/µL 裴在磅 (比裴/DNA = 3/1 瓦特/w) 和漩涡立即。在 RT 孵育10-15 分钟。</li>
		<li>从每个井中提取400µL 细胞培养基, 并将其添加到 DNA 培合剂中, 以中和 pH 值。将 DNA 混合物运球到细胞上。 注意:DMEM 通常含有苯酚红作为 pH 指标。在中和步骤中, 添加在管内的混合物的颜色将由黄色 (酸性) 变为红色 (中性)。虽然在酸性 ph 值下, 在 LBS 中形成 dna 复合体给出了最高的转染率50, 但 dna-裴复合物可以在 pH 值 7.4 (例如无血清 DMEM) 中直接形成。如果使用 DMEM 形成 DNA 络合物, 跳过中和步骤1.3.5。在任何情况下, 在形成复合物时, 混合物中不存在血清。</li>
	</ol>
</li>
	<li>收获细胞48小时后转染。<ol>
<li>吸入培养基和冲洗细胞一次与2毫升预热 PBS (37 °c)。添加800µL 的 PBS 补充0.5 毫米 EDTA 和孵化20分钟在37摄氏度。通过吹打来分离和暂停单元格。</li>
		<li>将细胞悬浮液转移到含有200µL PBS 的1.5 毫升管中, 辅以5毫米氯化镁2。</li>
		<li>离心机为2分钟, 在 800 x g, 并丢弃上清。 注意:协议可以在这里暂停。在这种情况下, 在液体 N2中将小球冻结, 并在-80 摄氏度处存储多达一个月。总是戴眼睛防护护目镜。</li>
	</ol>
</li>
	<li>添加100µL 三裂解缓冲 (50 毫米三盐酸 pH 8.0, 150 毫米氯化钠, 1% 海卫 X-100, 1 毫米 EDTA 和新鲜添加 PMSF) 到细胞丸和在冰上孵育30分钟。为了促进裂解, 漩涡每5分钟。</li>
	<li>在4摄氏度和 1.4万 x g 上旋转10分钟的细胞碎片, 将上清的90µL 转移到黑色96井板中. 使用带有荧光模块的板读取器测量 EGFP 和 mCherry 荧光。 注意:使用适当的励磁和发射过滤器 EGFP (λabs: 488 毫微米; λem: 509 毫微米) 和 mCherry (λabs: 588 毫微米; λem: 611 nm)。测量的 EGFP 值将跨越从模拟转染细胞获得的最小值和最大值之间的范围, 通常从野生型 EGFP 获得。注意在仪器中设置正确的测量窗口。</li>
	<li>计算了该方法的有效性, 以样品荧光与野生型 EGFP 表达的荧光比值为例。所有值都归一化为 mCherry 荧光。 <img alt="Equation 1" src="/files/ftp_upload/57069/57069eq1.jpg">
</li>
</ol>2. ncAAs 到受体中的遗传纳入配体-GPCR 相互作用的光交联映射 (图 3)
<ol><li>维持 HEK293T 细胞在 DMEM 补充与 10% (v/v) 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>种子细胞转染前的前一天。<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。淬火与10卷的完整培养基和并用重悬细胞通过吹打上下。使用 hemocytometer49计算挂起的单元格数。</li>
		<li>种子 5.0 x 105 293T 细胞每井在2毫升完全成长媒介在6井板材。对于每个要筛选的位置, 为绑定控制33,38准备1井每配体加一井。一个额外的井, 被转染野生类型 (重量) 受体可能被纳入检查的表达水平的突变体。</li>
	</ol></li>
	<li>后天, 控制汇合 (被细胞占据的区域) 在显微镜下。染细胞在70% 汇合使用裴。<ol>
<li>1h 在转染前, 将 Azi 添加到所有井中, 最终浓度为0.5 毫米。<ol>
<li>准备一个0.5 米 Azi 的库存解决方案。每 6-井板, 称1.2 毫克 Azi 入管子并且溶化它在15µL 0.5 M 氢氧化钠。稀释1.2 毫升完整介质中的库存溶液, 并将混合物的200µL 添加到每个井中。 注意:为每个实验准备一个 Azi 的新的库存解决方案。叠氮化物在水溶液中有很短的半衰期, 特别是在碱性 pH 值的情况下, AziRS 包含完好的, 但也含有退化的形态。</li>
		</ol>
</li>
		<li>染总金额为2µg DNA 每井: 1 µg 质粒编码的标志标记 GPCR 轴承的标签密码子在所需的位置和1µg 的质粒编码的正交对 Azi (E2AziRS51和4副本的同源抑制器-tRNA Bst山药)33,38。 注意:当包括一个量比较检查表达水平, 染的质粒 DNA 较低的重量受体。根据 GPCR 的不同, 0.2-0.5 µg 的质粒编码, 其受体产量与突变质粒的1.0 µg 相似。染在所有井中都有相同数量的 dna, 用模拟 (例如一个空向量) 填满丢失的 dna。</li>
		<li>按 1.3. 3-1. 3.5 的说明进行。</li>
		<li>48 h 转染后, 进行与步骤2.4 的照片交联的配体或转到步骤2.5 直接收获和分析验证受体表达。</li>
	</ol></li>
	<li>配体的照片交联。<ol>
<li>准备一个1,000x 配体库存溶液。将肽配体溶于亚砜中100µM 的浓度。 注意:配体浓度依赖于配体-GPCR 相互作用的离解常数 KD 。最后浓度为 100 x KD是可取的。如果肽配体是三氟乙酸酸 (TFA) 的盐, 则在计算分子量时考虑 TFA 的重量 (在肽中每基氨基酸的 1 x TFA)。此外, 考虑到肽是普遍吸湿。避免重复冷冻肽粉, 永远不要打开一个肽容器, 直到它没有达到室温。</li>
		<li>稀释配体储存液 1:1, 000 在包含5毫米 2-(12.5-羟乙基)-4-piperazineethanesulfonic 酸 (HEPES)-盐酸 pH 值 2, 1 毫米氯化钠的 HEPES 离解缓冲器 (HDB) 中, 由 0.1% BSA、0.01% 海卫 x 100、7.4 mm 氯化镁140组成的装订缓冲器和5毫米氯化钾, 每 Azi GPCR 的突变体准备1毫升。用1毫升的配体溶液取代细胞培养基。在 RT 孵化10分钟。 注意:调整孵化时间到特定 GPCR, 对配体动力学和受体内化进行核算。延长孵化时间不会提高交联率。</li>
		<li>在 365 nm 的 UV 交联剂中照射样品20分钟, 用 5 x 8 W 管和 ~ 5 厘米到细胞的距离。通过吹打分离细胞并将其转移到1.5 毫升的反应管中。在 800 x g 上将细胞颗粒3分钟, 然后丢弃上清。</li>
		<li>将蛋白酶抑制剂 (PI) 鸡尾酒片溶于1毫升25毫米 EDTA/小时2O 以制作50x 库存溶液。整除的 PI 库存解决方案, 并存储在-20 摄氏度。稀释50x 库存1:25 在 HDB 和并用重悬细胞小球在50µL 2 x PI 在 HDB。将液体中的单元格闪烁-冻结2。 注意:戴上护眼护目镜。在这一点上, 样品可以存储在摄氏-80 摄氏度, 长达一个月。继续执行步骤2.6。</li>
	</ol></li>
	<li>直接的细胞收获。<ol>
<li>吸入培养基。在 HDB 中加入800µL 0.5 毫米 EDTA。在 RT 或冰上孵化10分钟。</li>
		<li>通过吹打和向下分离细胞, 并将它们转化为1.5 毫升的反应管。在 HDB 中添加200µL 5 mM 氯化镁2 。在 800 x g 上将细胞颗粒3分钟, 然后丢弃上清。</li>
		<li>并用重悬细胞颗粒在50µL 2 x PI 在 HDB 和闪光冷冻在液体N2。戴上护眼护目镜。 注意:在这一点上, 样品可以储存在-80 摄氏度, 长达1月。</li>
	</ol></li>
	<li>细胞裂解。<ol>
<li>在水浴中解冻细胞在37°c 为 30-45 s 和漩涡简要地。从现在起保持样品的冷。颗粒膜在 2500 x g 和4°c 为10分钟丢弃上清, 其中含有大量胞浆蛋白。</li>
		<li>并用重悬50µL HEPES 裂解缓冲液中含有50毫米 HEPES 盐酸 pH 7.5, 150 毫米氯化钠, 10% 甘油, 1% 海卫 X-100, 1.5 毫米氯化镁2, 1 毫米 EGTA, 1 毫米的微丸和新鲜添加 2 X PI 鸡尾酒。完全混合。每5分钟溶解30分钟的冰和涡旋细胞。</li>
		<li>在 1.4万 x g 和4摄氏度下, 将细胞碎片向下旋转10分钟。立即将上清液转移到新的反应管上。 注意:立即进行分析。裂解物可以储存在摄氏-20 摄氏度, 但是, 每一个冻融周期都会损害结果的质量。</li>
	</ol></li>
	<li>西方污点分析。<ol>
<li>要准备样品, 取3-5 µL 裂解液并将其填满7µL 与 H2o 添加2µL 1 M 和3µL 4 x 样本缓冲区, 其中包含63毫米三盐酸 pH 6.8, 2% SDS, 10% 甘油和0.04% 溴酚蓝色。孵育30分钟, 在37摄氏度。</li>
		<li>当 GPCR 是糖化和微弱或沾污带是一个问题, deglycosylate 样品与 PNGase F 增加信号强度和锐化波段。根据供应商的协议, 在10µL 的总容积中使用3-5 µL 裂解液和 deglycosylate。添加3µL 4 x 示例缓冲区。 注意:膜蛋白在多个部位和状态下通常是糖化的, 这会损害 SDS 页分析中分辨率的质量。但是, 不要 deglycosylate 样本来分析 Azi GPCR 突变体的表达水平, 因为它与评估细胞表面完全糖化、成熟受体的部分有关。</li>
		<li>通过标准的 SDS 页和印迹转移蛋白, 通过 PVDF 膜解决样品。 警告:丙烯酰胺是神经毒性。戴上手套, 保护眼睛。</li>
		<li>在4摄氏度的5% 脱脂牛奶中, 用20毫米三盐酸 pH 7.4、0.15 米氯化钠和0.1% 吐温 20, 将1小时的膜阻挡在 RT 或隔夜的室温下。</li>
		<li>用抗配体抗体对膜进行探针-共轭二次抗体。在与 tb 之间进行清洗。为了检测 Azi-GPCR 的表达水平, 用商用的 HRP 抗体探测膜 (参见材料表)。</li>
		<li>使用自制的 ECL 试剂进行增强化学发光 (ecl) 反应, 在黑暗中检测信号5分钟。</li>
	</ol></li>
</ol>3. 活哺乳动物细胞受体的超快 Bioorthogonal 标记
注意:该协议针对4井腔盖玻片 (井区 = 2.2厘米2) 进行了优化。对于不同的大小, 协议必须相应地进行缩放。
<ol><li>显微镜切片表面涂层。在无菌罩下进行全过程。<ol>
<li>在50毫米硼酸盐缓冲器 (pH 8.5) 的浓度为1毫克/毫升, 制备聚 d-赖氨酸氢溴酸盐 (MW=500-550 kDa) (PDL) 股票溶液。贮存在4摄氏度, 可达6月。不要冻结。</li>
		<li>稀释的 PDL 股票解决方案1:40 在无菌的超纯水到最终浓度的25µg/毫升 (工作解决方案), 然后过滤解决方案通过0.22 µm 无菌过滤器。 注意:工作解决方案可存储在4摄氏度, 长达3月。</li>
		<li>用500µL 的 PDL 工作解决方案, 充分覆盖显微镜下各井的底部。孵育20分钟在 RT 和吸入的 PDL 工作解决方案。 注意:PDL 工作解决方案最多可使用三次。如果需要重新使用该解决方案, 请将所用的解决方案从涂层的幻灯片转移到新鲜的无菌管, 并相应地给管子贴上标签。切勿将再生溶液与新鲜溶液混合使用。</li>
		<li>用700µl 无菌的超纯水冲洗每个井 3 x, 让干燥至少1小时。 注意:准确冲洗井是非常重要的, 因为 PDL 溶液的残留物对细胞是有毒的。涂层的幻灯片可以直接使用显微镜或存储长达一周的4摄氏度。</li>
	</ol>
</li>
	<li>维持 HEK293T 细胞在 DMEM 补充与 10% (v/v) 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>种子细胞转染前的前一天。<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卷的完全培养基淬火, 并通过吹打并用重悬细胞。使用 hemocytometer49计算挂起的单元格数。</li>
		<li>种子 1.0 x 105 HEK293T 细胞每井 (区域 2.2 cm²) 在600µL 染料自由完全 DMEM。 注意:为成像目的, 从一开始就不含任何染料的介质中工作是非常方便的。染料自由 DMEM 配方是商业上可用的。</li>
	</ol>
</li>
	<li>控制汇流 (被细胞占据的区域) 在显微镜下, 用脂质为基础的转染试剂染70% 汇合的细胞。<ol>
<li>1 h 在转染前, 在0.2 米氢氧化钠和 15% (v/v) 亚砜上准备一个新的100毫米的 TCO * K 的库存解决方案。</li>
		<li>每井, 混合3µl 的 TCO * K 库存解决方案与12µl 1 米 HEPES pH 7.4。将解决方案轻轻地添加到井中, 最终拥有 TCO * K 浓度为0.5 毫米。</li>
		<li>每井准备500的 DNA。在离心管中, 稀释 200 pcDNA3.1_CRF1R-95 标记 EGFP, 200 ng 的质粒编码为MbPylRSAF/tRNAPyl正交对 (四盒 tRNAM15) 和 100 ng pcDNA3.1_Arrestin3质粒在50µL 培养基 (无染料, 无血清, 无抗生素)。 注意:一般而言, Arrestin 的联合转染没有必要观察 GPCR 内化。然而, 转染 Arrestin3 加速了 CRF1R 的内化, 这在分析许多突变体的内化时非常方便。</li>
		<li>稀释1.25 µL 脂基转染试剂 (2.5 µL 每1µg 的 DNA) 在50µL 培养基 (无染料, 无血清, 无抗生素), 并添加溶液的 dna 混合物。漩涡立即和孵化5-10 分钟在 RT. 将 DNA 脂质复合物添加到细胞中。 注意:根据我们的经验, 以脂质为基础转染的细胞的形态学看起来比与裴转基因的细胞更具生理性。由于裴给出更高的转染效率, 裴应该是首选的下游应用, 如西方印迹, 而脂质的转染是一个更好的选择染细胞进行成像实验。</li>
	</ol>
</li>
	<li>24 h 后转染, 标签的受体与荧光染料。<ol>
<li>在超纯 H2O 中, 在亚砜和10毫克/毫升 DNA 染色染料库存溶液中制备0.5 毫米染料四嗪类溶液。</li>
		<li>将100µL 介质从每个井转移到1.5 毫升的反应管中。添加1.8 µL 的染料四嗪类溶液和0.3 µL 的 DNA 染色染料库存溶液。将含有染料的培养基转回到井中, 在37摄氏度时孵育5分钟。 注意:四嗪类橙荧光染料的最终浓度为1.5 µM。</li>
		<li>吸入培养基, 用 PBS 轻轻冲洗两次细胞, 以去除过量的染料。添加600µL 完全染料游离生长培养基预热到37摄氏度。</li>
	</ol>
</li>
	<li>荧光显微镜和受体内化。<ol>
<li>使用适合于 GFP (λabs的滤镜, 可视化 63x (或类似) 放大倍数下的标记受体: 488 毫微米; λem: 509 nm), 橙色荧光染料 (λabs: 550 nm; λem: 570 nm) 和 DNA 染色染料 (λabs: 350 毫微米;λem: 461 nm)。在激活受体之前, 用每个过滤器拍照。</li>
		<li>用 200 nM 的 Ucn1 促进受体内部化。<ol>
<li>在亚砜中制备200µM 的 1000 x Ucn1 库存溶液。 注意:根据肽的溶解度, 你可以在纯净的水或缓冲中准备存货。</li>
			<li>将100µL 培养基从井中转移到1.5 毫升反应管中, 加入0.6 µL 的肽激动剂库存溶液。把介质移回井中。</li>
			<li>观察显微镜下的内部化。使用前面提到的过滤器, 在清楚发现的内部化发生 (10-15 分钟到小时, 取决于 Arrestins 的受体和过度表达) 之后拍照。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

作者没有要声明的冲突。

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><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</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)&amp;nbsp;</td><td>home-made</td><td></td><td>10 mg/l luminol in 0.1 M Tris-HCl pH 8.6 ; 1100 mg/l&amp;nbsp; p-coumaric acid in DMSO ; 30&amp;nbsp;% H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; (1,000 : 100 : 0.3)</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&amp;nbsp;</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>MgCl&lt;sub&gt;2&lt;/sub&gt;</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&amp;uuml;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&amp;nbsp;</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>&lt;strong&gt;Cell lines&lt;/strong&gt;</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>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp;</td><td>BMG LABTECH</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</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&amp;nbsp;</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>&lt;strong&gt;Antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-FLAG-HRP M2 antibody conjugate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A8592&amp;nbsp;</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 &lt;br/&gt;Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)</td></tr><tr><td>Rabbit-anti-Ucn1 antibody</td><td>home-made</td><td>PBL #5779</td><td>polyclonal &lt;br/&gt;Turnbull, A.V., Vaughan, J., Rivier, J.E., and Vale, W.W. Endocrinology, 140, (1), 71-78 (1999)</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 ...

optimizing-genetic-incorporation-chemical-probes-into-gpcrs-for-photo

Research

JoVE Journal

Chemistry

8.4K 次观看.  University of Leipzig. 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。

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

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.

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 ...

科研

生物化学

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

遗传学

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

Interactions between G-protein coupled receptors (GPCRs) and &#946;-arrestins are vital processes with physiological implications of great importance. Currently, the characterization of novel drugs towards their interactions with &#946;-arrestins and other cytosolic proteins is extremely valuable in the field of GPCR drug discovery particularly during the study of GPCR biased agonism. Here, we show the application of a novel structural complementation assay to accurately monitor receptor-&#946;-arrestin interactions in real time living systems. This method is simple, accurate and can be easily extended to any GPCR of interest and also it has the advantage that it overcomes unspecific interactions due to the presence of a low expression promoter present in each vector system. This structural complementation assay provides key features that allow an accurate and precise monitoring of receptor-&#946;-arrestin interactions, making it suitable in the study of biased agonism of any GPCR system as well as GPCR c-terminus &#8216;phosphorylation codes&#8217; written by different GPCR-kinases (GRKs) and post-translational modifications of arrestins that stabilize or destabilize the receptor-&#946;-arrestin complex.

GPCR-&#946;-arrestin interactions are an emerging field in GPCR drug discovery. Accurate, precise and easy to set up methods are necessary to monitor such interactions in living systems. We show a structural complementation assay to monitor GPCR-&#946;-arrestin interactions in real time living cells, and it can be extended to any GPCR.

监测GPCR-β-阻丁1/2实时生命系统中的相互作用,加速药物发现

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

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 ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

用于重组G蛋白偶联受体高通量筛选的"双加法"钙荧光测定

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

在体内 G蛋白偶联受体相互作用光谱分辨的双光子显微镜定量

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

生物学

Imaging G-protein Coupled Receptor (GPCR)-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1. This guided cell migration is referred as chemotaxis, which is essential for various cells to carry out their functions such as trafficking of immune cells and patterning of neuronal cells 2, 3. A large family of G-protein coupled receptors (GPCRs) detects variable small peptides, known as chemokines, to direct cell migration in vivo 4. The final goal of chemotaxis research is to understand how a GPCR machinery senses chemokine gradients and controls signaling events leading to chemotaxis. To this end, we use imaging techniques to monitor, in real time, spatiotemporal concentrations of chemoattractants, cell movement in a gradient of chemoattractant, GPCR mediated activation of heterotrimeric G-protein, and intracellular signaling events involved in chemotaxis of eukaryotic cells 5-8. The simple eukaryotic organism, Dictyostelium discoideum, displays chemotaxic behaviors that are similar to those of leukocytes, and D. discoideum is a key model system for studying eukaryotic chemotaxis. As free-living amoebae, D. discoideum cells divide in rich medium. Upon starvation, cells enter a developmental program in which they aggregate through cAMP-mediated chemotaxis to form multicullular structures. Many components involved in chemotaxis to cAMP have been identified in D. discoideum. The binding of cAMP to a GPCR (cAR1) induces dissociation of heterotrimeric G-proteins into G&gamma; and G&beta;&gamma; subunits 7, 9, 10. G&beta;&gamma; subunits activate Ras, which in turn activates PI3K, converting PIP2 into PIP3 on the cell membrane 11-13. PIP3 serve as binding sites for proteins with pleckstrin Homology (PH) domains, thus recruiting these proteins to the membrane 14, 15. Activation of cAR1 receptors also controls the membrane associations of PTEN, which dephosphorylates PIP3 to PIP2 16, 17. The molecular mechanisms are evolutionarily conserved in chemokine GPCR-mediated chemotaxis of human cells such as neutrophils 18. We present following methods for studying chemotaxis of D. discoideum cells. 1. Preparation of chemotactic component cells. 2. Imaging chemotaxis of cells in a cAMP gradient. 3. Monitoring a GPCR induced activation of heterotrimeric G-protein in single live cells. 4. Imaging chemoattractant-triggered dynamic PIP3 responses in single live cells in real time. Our developed imaging methods can be applied to study chemotaxis of human leukocytes.

Imaging G-protein Coupled Receptor GPCR-mediated Signaling Events that Control Chemotaxis of Dictyostelium Discoideum

Here, we describe detailed live cell imaging methods for investigating chemotaxis. We present fluorescence microscopic methods to monitor spatiotemporal dynamics of signaling events in migrating cells. Measurement of signaling events permits us to further understand how a GPCR-signaling network achieves gradient sensing of chemoattractants and controls directional migration of eukaryotic cells.

成像摹蛋白偶联受体（GPCR）介导的信号，控制趋化粘菌</em

Many eukaryotic cells can detect gradients of chemical signals in their environments and migrate accordingly 1.  This guided cell migration is referred as ...

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

生物工程

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

基因编码的分子探针来研究G蛋白偶联受体

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

This protocol describes confocal microscopy detection of G protein-coupled receptor (GPCR) internalization in mammalian cells. It includes the basic cell culture, transfection, and confocal microscopy procedure and provides an efficient and easily interpretable method to detect the subcellular localization and internalization of fusion-expressed GPCR.

配体激活的G蛋白偶联受体内在化的检测通过共聚焦显微镜

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent ...