Abstract
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.
Highlights
The new class of NIR fluorescent proteins (FPs) was developed from the subclass of phytochromes, called bacteriophytochromes (BphPs)[9,10,11,12,13,14] Phytochromes constitute a family of light sensors in plants, fungi and bacteria[15 ]; their light-sensing module comprises, so-called PAS, GAF and PHY domains
In iRFP702, the fluorescence lifetime increased to 1.35 ns (Fig. 2a, open circles and blue line), corresponding to a kinetic isotope effect (KIE) of 1.8
Such sub-ns lifetimes are consistent with their fluorescence quantum yields of 7–8%14 and indicate a BV inherent radiative lifetime of 9.5 ns, which is somewhat longer than that estimate of Toh et al for RpBphP3 (7.5 ns)[28]
Summary
The new class of NIR FPs was developed from the subclass of phytochromes, called bacteriophytochromes (BphPs)[9,10,11,12,13,14] Phytochromes constitute a family of light sensors in plants, fungi and bacteria[15 ]; their light-sensing module comprises, so-called PAS, GAF and PHY domains. Note that this structure provides only an approximate structural model for the iRFPs as various residues in the vicinity of BV were altered[9,14]. The iRFP proteins efficiently utilize an endogenous BV chromophore of mammalian cells and tissues to become brightly fluorescent, which is in contrast to other reported NIR FPs that requires adding of an excess of external BV to acquire fluorescence[10]. Molecular knowledge of excited-state and reaction intermediate dynamics will contribute to a comprehensive picture of BphPs and iRFPs fluorescence function, and may serve as a basis for further rational engineering of advanced NIR FPs from BphPs
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