Heterotrimeric G-proteins (Gαβγ) are the main transducers of signals from GPCRs, mediating the action of countless natural stimuli and therapeutic agents. However, there are currently no robust approaches to directly measure the activity of endogenous G-proteins in cells. Here, we describe a suite of optical biosensors that detect endogenous active G-proteins with sub-second resolution in live cells. Using a modular design principle, we developed genetically encoded, unimolecular biosensors for endogenous Gα-GTP and free Gβγ: the two active species of heterotrimeric G-proteins. This design was leveraged to generate biosensors with specificity for different heterotrimeric G-proteins or for other G-proteins, such as Rho GTPases. Versatility was further validated by implementing the biosensors in multiple contexts, from characterizing cancer-associated G-protein mutants to neurotransmitter signaling in primary neurons. Overall, the versatile biosensor design introduced here enables studying the activity of endogenous G-proteins in live cells with high fidelity, temporal resolution, and convenience.

异三聚体 G 蛋白 (Gαβγ) 是 GPCR 信号的主要传递者，介导无数天然刺激物和治疗剂的作用。然而，目前没有可靠的方法来直接测量细胞中内源性 G 蛋白的活性。在这里，我们描述了一套光学生物传感器，可在活细胞中以亚秒级分辨率检测内源性活性 G 蛋白。使用模块化设计原理，我们为内源性 Gα-GTP 和游离 Gβγ（异源三聚体 G 蛋白的两种活性物质）开发了基因编码的单分子生物传感器。这种设计被用来生成对不同异源三聚体 G 蛋白或其他 G 蛋白（如 Rho GTPases）具有特异性的生物传感器。通过在多种环境中实施生物传感器，进一步验证了多功能性，从表征癌症相关的 G 蛋白突变体到初级神经元中的神经递质信号。总的来说，这里介绍的多功能生物传感器设计能够以高保真度、时间分辨率和便利性研究活细胞中内源性 G 蛋白的活性。