Ultrafast excited-state dynamics and fluorescence deactivation of near-infrared fluorescent proteins engineered from bacteriophytochromes

Near-infrared fluorescent proteins, iRFPs, are recently developed genetically encoded fluorescent probes for deep-tissue in vivo imaging. Their functions depend on the corresponding fluorescence efficiencies and electronic excited state properties. Here we report the electronic excited state deactivation dynamics of the most red-shifted iRFPs: iRFP702, iRFP713 and iRFP720. Complementary measurements by ultrafast broadband fluorescence and absorption spectroscopy show that single exponential decays of the excited state with 600 ∼ 700 ps dominate in all three iRFPs, while photoinduced isomerization was completely inhibited. Significant kinetic isotope effects (KIE) were observed with a factor of ∼1.8 in D₂O, and are interpreted in terms of an excited-state proton transfer (ESPT) process that deactivates the excited state in competition with fluorescence and chromophore mobility. On this basis, new approaches for rational molecular engineering may be applied to iRFPs to improve their fluorescence.