Abstract
Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. However, the most fundamental property underpinning photooxidation, the electron detachment energy, has only been measured for the deprotonated GFP chromophore in the gas phase. Here, we use multiphoton ultraviolet photoelectron spectroscopy in a liquid-microjet and high-level quantum chemistry calculations to determine the electron detachment energy of the GFP chromophore in aqueous solution. The aqueous environment is found to raise the detachment energy by around 4 eV compared to the gas phase, similar to calculations of the chromophore in its native protein environment. In most cases, electron detachment is found to occur resonantly through electronically excited states of the chromophore, highlighting their importance in photo-induced electron transfer processes in the condensed phase. Our results suggest that the photooxidation properties of the GFP chromophore in an aqueous environment will be similar to those in the protein.
Highlights
Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions
The first absorption bands in the electronic absorption spectra of GFP and the isolated p-HBDI− chromophore in vacuo lie remarkably close to one another[11,12,13,14,15], suggesting that the β-barrel structure provides an electronic environment that is similar to a vacuum; the environment of the chromophore plays a crucial role in defining the electronic relaxation dynamics following photoexcitation of the first electronically excited state
Our results suggest that the photooxidation properties of the deprotonated GFP chromophore in aqueous solution are similar to those of the chromophore in its natural protein environment
Summary
Green fluorescent protein (GFP), the most widely used fluorescent protein for in vivo monitoring of biological processes, is known to undergo photooxidation reactions. The lack of fluorescence from the free chromophore at physiological temperatures has been attributed to ultrafast double-bond isomerisation, followed by internal conversion back to the electronic ground state[19,20], with timescales that are similar in the gas phase[21] and in solution[19,20], suggesting that the intrinsic electronic properties of the chromophore determine the relaxation mechanism. The electron detachment energy, the most fundamental property underpinning photooxidation, and the electron binding energies of the higher lying electronic states have only been determined experimentally for the deprotonated GFP chromophore in the gas phase[11,13,21,24,25,26,27,28]. Our results suggest that the photooxidation properties of the deprotonated GFP chromophore in aqueous solution are similar to those of the chromophore in its natural protein environment
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