使用单分子FRET可视化膜受体的构象动力学
Summary
本研究提出了一种详细的程序，通过非天然氨基酸（UAA）掺入 使用 位点特异性标记对G蛋白偶联受体（GPCR）进行单分子荧光共振能量转移（smFRET）实验。该协议为 smFRET 样品制备、实验和数据分析提供了分步指南。
Abstract
细胞对外部信号做出反应的能力对于细胞发育、生长和存活至关重要。为了响应来自环境的信号，细胞必须能够识别和处理它。这项任务主要依靠膜受体的功能，膜受体的作用是将信号转化为细胞的生化语言。G蛋白偶联受体（GPCR）是人类最大的膜受体蛋白家族。在GPCR中，代谢性谷氨酸受体（mGluR）是一个独特的亚类，其功能是专性二聚体，并具有包含配体结合位点的大细胞外结构域。mGluR结构研究的最新进展提高了对其活化过程的理解。然而，在激活和调制过程中通过mGluR传播大规模构象变化知之甚少。单分子荧光共振能量转移（smFRET）是一种在单蛋白水平上可视化和量化生物分子结构动力学的强大技术。为了可视化mGluR2活化的动态过程，开发了基于非天然氨基酸（UAA）掺入的荧光构象传感器，该传感器允许位点特异性蛋白质标记而不会干扰受体的天然结构。此处描述的协议解释了如何执行这些实验，包括新型UAA标记方法，样品制备以及smFRET数据采集和分析。这些策略是可推广的，可以扩展到研究各种膜蛋白的构象动力学。
Introduction
跨质膜的信息传递在很大程度上取决于膜受体的功能1。配体与受体结合导致构象变化和受体活化。这个过程在本质上通常是变构的2.G蛋白偶联受体（GPCR）拥有800多个成员，是人类最大的膜受体家族3。由于其在几乎所有细胞过程中的作用，GPCR已成为治疗开发的重要靶标。在GPCR信号传导的典型模型中，激动剂激活导致受体的构象变化，随后通过在Gα的核苷酸结合口袋中将GDP交换为GTP来激活异源三聚体G蛋白复合物。活化的Gα-GTP和Gβγ亚基然后控制下游效应蛋白的活性并传播信号级联4，5。这种信号传导过程基本上取决于配体改变受体三维形状的能力。对配体如何实现这一目标的机制理解对于开发新疗法和设计合成受体和传感器至关重要。
代谢性谷氨酸受体（mGluRs）是C类GPCR家族的成员，对于谷氨酸的缓慢神经调节作用和调节神经元兴奋性很重要6，7。在所有GPCR中，C类GPCR在结构上是独一无二的，因为它们作为专性二聚体起作用。mGluR包含三个结构域：金星捕蝇器（VFT）结构域，富含半胱氨酸的结构域（CRD）和跨膜结构域（TMD）8。活化过程中的构象变化是复杂的，涉及在12 nm距离上传播的局部和全局构象耦合，以及二聚体的协同性。中间构象、状态的时间顺序和状态之间的转换速率是未知的。通过实时跟踪单个受体的构象，可以识别瞬时中间状态和激活过程中构象变化的序列。这可以通过应用单分子荧光共振能量转移9，10（smFRET）来实现，就像最近应用于可视化mGluR211活化过程中构象变化的传播一样。FRET实验的一个关键步骤是通过将供体和受体荧光团插入目标蛋白质的位点特异性插入来生成FRET传感器。采用非天然氨基酸（UAA）掺入策略12，13，14，15，以克服典型的位点特异性荧光标记技术的局限性，该技术需要创建无半胱氨酸突变体或插入大型基因编码标签。这允许观察到基本的致密变构接头的构象重排，该接头连接了mGluR2的配体结合和信号结构域。在该协议中，提供了在mGluR2上进行smFRET实验的分步指南，包括使用UAA对mGluR2进行位点特异性标记以使用铜催化叠氮化物环化反应附着荧光团的方法。此外，该协议描述了直接捕获膜蛋白和数据分析的方法。此处概述的方案也适用于研究其他膜蛋白的构象动力学。
Protocol
该协议的整体工作流程如图 1 所示。 1. 样品室的准备 玻片和盖玻片清洁注意：这些步骤旨在清洁载玻片的表面以及盖玻片，并为氨基硅烷化做好准备。对表面束缚分子进行单分子荧光实验的一个关键要求是钝化表面。最可靠和可重复的钝化技术涉及将惰性聚合物链作为致密层共价附着到玻璃表面。聚乙二醇（PEG）是用于表面钝化的最有效?…
Representative Results
基于UAA的FRET传感器的表达和荧光标记本文讨论了在mGluR2（548UAA）的CRD内插入和荧光标记UAA（AZP）的示例性结果11。如前所述，要将AZP插入mGluR2中，工程翻译机制的共表达是必要的，其中包括修饰的tRNA合成酶和互补tRNA（pIRE4-Azi），以及使用诱变创建的在位置548处含有琥珀色密码子的mGluR2（图2A，B）。通过铜催化的环加成反应（<st…
Discussion
GPCR是在细胞膜上起作用以启动信号转导的蛋白质。许多GPCR由多个域组成，信令依赖于域之间的合作相互作用。为了调节这些膜受体的性质，必须了解多个结构域的动态行为。单分子荧光共振能量转移（smFRET）是一种荧光技术，能够实时测量蛋白质构象和动力学11，32。这里描述了一种结合smFRET，单分子下拉（SiMPull）和全内反射荧光（T…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢Reza Vafabakhsh实验室成员的讨论。这项工作得到了美国国立卫生研究院拨款R01GM140272（R.V.），西北大学Searle生命科学领导基金和芝加哥生物医学联盟的支持，并得到了芝加哥社区信托基金的Searle基金（对R.V.）的支持。B.W.L.得到了美国国家普通医学科学研究所（NIGMS）培训补助金T32GM-008061的支持。
Materials
| (+)-Sodium L-Ascorbate | Sigma Aldrich | Cat # 11140-250G | |
| 4-azido-L-phenylalanine | Chem-Impex International | Cat # 06162 | |
| 548UAA | Liauw et al. 2021 | Transfected construct | |
| Acetic Acid | Fisher Chemical | 64-19-7 | |
| Acetone | Fisher Chemical | 67-64-1 | |
| Adobe Illustrator (2022) | https://www.adobe.com/ | RRID:SCR_010279 | Software, algorithm |
| Aminoguanidine (hydrochloride) | Cayman Chemical | 81530 | |
| Aminosilane | Aldrich | 919-30-2 | |
| Bath Sonicator 2.8 L | Fisher Scientific | Ultrasonic Bath 2.8 L | |
| Biotin-PEG | Laysan Bio Inc | Item# Biotin-PEG-SVA-5000-100mg | |
| BTTES | Click Chemistry Tools | 1237-500 | |
| Copper (II) sulfate | Sigma Aldrich | Cat # 451657-10G | |
| Cover slip | VWR | 16004-306 | Sample chamber |
| Cy3 Alkyne | Click Chemistry Tools | TA117-5 | |
| Cy5 Alkyne | Click Chemistry Tools | TA116-5 | |
| DDM | Anatrace | Part# D310 1 GM | Detergent |
| DDM-CHS (10:1) | Anatrace | Part# D310-CH210 1 ML | Detergent with cholecterol |
| Defined Fetal Bovine Serum | Thermo Fisher Scientific | SH30070.03 | |
| Di01-R405/488/561/635 | Semrock | Notch filter | |
| DMEM | Corning | 10-013-CV | |
| EMCCD | Andor | DU-897U | Camera |
| ET542lp | Chroma | Long pass emission filter | |
| FF640-FDi01 | Semrock | Emission dichroic filter | |
| FLAG-tag antibody | Genscript | A01429 | |
| Fluorescent bead | Invitrogen T7279 | TetraSpeck microspheres | Spherical bead |
| Glass slides | Fisherfinest | 12-544-4 | sample chamber |
| Glutamate | Sigma Aldrich | Cat # 6106-04-3 | |
| HEK 293T | Sigma Aldrich | Cat # 12022001 | Cell line |
| HEPES | FisherBioReagents | 7365-45-9 | |
| Image splitter | OptoSplit II | ||
| KOH | Fluka | 1310-58-3 | |
| Laser | Oxxius | 4-line laser combiner | |
| Lipofectamine 3000 Transfection Reagent | Thermo Fisher Scientific | L3000015 | Transfection Reagent |
| Methanol | Fisher Chemical | 67-56-1 | |
| Microscope | Olympus | Olympus IX83 | |
| Milli-Q water | Barnstead | Water Deionizer | |
| m-PEG | Laysan Bio Inc | Item# MPEG-SIL-5000-1g | |
| NF03-405/488/532/635 | Semrock | Dichroic mirror | |
| OptiMEM | Thermo Fisher Scientific | 51985091 | Reduced Serum Medium |
| OptiMEM/Reduced serum medium | Thermo Fisher Scientific | ||
| OriginPro (2020b) | https://www.originlab.com/ | RRID:SCR_014212 | Data analysis and graphing software |
| Penicillin-Streptomycin | Gibco | 15140-122 | |
| pIRE4-Azi | Addgene | Plasmid # 105829 | Transfected construct |
| Poly-L-lysine hydrobromide | Sigma Aldrich | Cat # P2636 | |
| Protocatechuic acid (PCA) | HWI group | 99-50-3 | |
| smCamera (Version 1.0) | http://ha.med.jhmi.edu/resources/ | Camera software | |
| Sodium bicarbonate | FisherBioReagents | 144-55-8 | |
| Sodium hydroxide (NaOH) | Sigma | 1310-73-2 | |
| Syringe filter | Whatman UNIFLO | Cat#9914-2502 | Liquid filtration |
| Trolox | Sigma | 53188-07 |
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Cite This Article
Banerjee, C., Liauw, B. W., Vafabakhsh, R. Visualizing the Conformational Dynamics of Membrane Receptors Using Single-Molecule FRET. J. Vis. Exp. (186), e64254, doi:10.3791/64254 (2022).