神经元活性和细胞内信号转导是神经环路可塑性的重要基础。而基于环化重排荧光蛋白（cpFP）探针的高时空分辨率活体双光子成像是研究上述两种过程的有力工具。因此，开发关键信号分子的高性能探针非常迫切。cAMP是G蛋白偶联受体的关键第二信使，在突触可塑性调控方面具重要作用。然而，现有的cAMP cpFP探针在37°C培养细胞中存在动态范围小（ΔF/F0<1.5）和荧光亮度低的问题, 无法满足活体成像要求。为克服上述缺点，申请人研发了新的动态范围较大的（ΔF/F0=4）cAMP cpFP探针G-Flamp0.5，且解析了其晶体结构。本项目拟结合晶体结构介导的理性设计和筛选技术，将其发展为大动态范围（ΔF/F0~10）、高亮度和高特异性的高性能探针，并将其用于活体双光子成像。以上目标的实现不仅可提供活体cAMP信号时空动态的信息，也能加深理解探针结构与性能间关系，为开发其它cpFP探针提供坚实的基础。

Neuronal activity and intracellular signaling transduction are crucial for neuronal circuit plasticity. In vivo two-photon imaging along with circularly permuted fluorescent protein (cpFP)-based indicators is a powerful technique for monitoring the two processes with high spatial and temporal resolution. Hence, it’s highly desirable to develop high-performance indicators for key signaling molecules. Cyclic AMP (cAMP) is a key second messenger that integrates signals from the majority of GPCRs and plays a crucial role in synaptic plasticity. However, current cAMP cpFP sensors suffer from small dynamic range (ΔF/F0<1.5) and low brightness in cells cultured at 37°C, making in vivo two-photon cAMP imaging being challenging. To overcome these limitations, we engineered a new cAMP cpFP sensor named G-Flamp0.5 with a ΔF/F0 of 4 and resolved its crystal structure. In this project, we will use a combination of structure-guided rational design and library screening to develop high-performance G-Flamp0.5-based cAMP sensors with a larger dynamic range (ΔF/F0~10), higher brightness and high specificity, and apply them to in vivo two-photon imaging. Progresses in these aims will not only provide new information about the spatial-temporal dynamics of cAMP signaling in vivo, but also obtain a deeper understanding of the relationship between the structure and properties of the sensors and facilitate the development of other cpFP indicators.