Substituent effects on the photophysics of the kaede chromophore

Kaede is the prototype of the optical highlighter proteins, which are an important subclass of the fluorescent proteins that can be permanently switched from green to red emitting forms by UV irradiation. This transformation has important applications in bioimaging. Optimising brightness, i.e. enhancing fluorescence characteristics, in these proteins is an important objective. At room temperature, the excited state dynamics of the red form of the kaede chromophore are dominated by a broad distribution of conformers with distinct excited state kinetics. Here, we investigate substituent effects on the photophysics of this form of the kaede chromophore. While an electron withdrawing substituent (nitro) red shifts the electronic spectra, the modified chromophores showed no significant solvatochromism. The lack of solvatochromism suggests small changes in permanent dipole moment between ground and excited electronic states, which is consistent with quantum chemical calculations. Ultrafast fluorescence and transient absorption spectroscopy reveal correlations between radiative and nonradiative decay rates of different conformers in the chromophores. The most significant effect of the substituents is to modify the distribution of conformers. The results are discussed in the context of enhancing brightness of optical highlighter proteins. Ultrafast time resolved spectroscopy probes substituent dependent photophysics of the kaede fluorescent protein chromophore.