Sensitive detection via the time-resolved fluorescence of circularly permuted yellow fluorescent protein biosensors

Circularly permuted fluorescent proteins (cpFP) have been used to develop diverse biosensors for monitoring cellular metabolites and states. In this article, we applied time-resolved spectroscopy to study a novel genetically encoded histidine biosensor, FHisJ, in vitro and in vivo. The average lifetime excited at 485 nm changes by 1.2 ns upon binding of histidine, which is much wider than most cpYFP-based biosensors. The ratio of fractional intensities changes 8-fold, which is higher than the excitation ratio measured at same conditions. More importantly, we study the photocycles of various cpYFP biosensors, i.e, FHisJ, SoNar, Frex and iNap. This work studies the difference of energy levels of cpYFP biosensors before and after binding with analytes, and reveals its effect on fluorescence intensity and lifetime. The cpYFP biosensors exist in 3 forms (A, I and B), but the fluorescence mainly comes from the excited states I* and B*. The dynamic range of the excitation ratio depends on the variation of A/(I+B) to some extent. However, the dynamic range of time-resolved fluorescence is related to the variation of I*/B*. This study will inspire the design of novel cpFP biosensors, especially those based on time-resolved fluorescence.