The green fluorescent protein (GFP) is employed extensively as a marker in biology and the life sciences as a result of its spectacular fluorescence properties. Here, we employ femtosecond time-resolved photoelectron spectroscopy to investigate the ultrafast excited state dynamics of the isolated GFP chromophore anion. Excited state population is found to decay bi-exponentially, with characteristic lifetimes of 330 fs and 1.4 ps. Distinct photoelectron spectra can be assigned to each of these timescales and point to the presence of a transient intermediate along the decay coordinate. Guided by ab initio calculations, we assign these observations to twisting about the CC -C bridge followed by internal conversion to the anion ground state. The dynamics in vacuo are very similar to those observed in solution, despite the difference in absorption spectra between the two media. This is consistent with the protein environment restricting rotation about the CC -C bond in order to prevent ultrafast internal conversion and preserve the fluorescence.

By uncovering the mechanism of UV resonant electron emission, we show that the GFP biomimetics are more stable to photooxidation.

Isolated model anion chromophores of the green and cyan fluorescent proteins were generated in an electrospray ion source, and their photodetachment spectra were recorded using photoelectron imaging. Vertical photodetachment energies of 2.85(10) and 4.08(10) eV have been measured for the model green fluorescent protein chromophore anion, corresponding to photodetachment from the ground electronic state of the anion to the ground and first excited electronic states of the radical, respectively. For the model cyan fluorescent protein chromophore anion, vertical photodetachment energies of 2.88(10) and 3.96(10) eV have been measured, corresponding to detachment from the ground electronic state of the anion to the ground and first excited electronic states of the neutral radical, respectively. We also find evidence suggesting that autoionization of electronically excited states of the chromophore anions competes with direct photodetachment. For comparison and to benchmark our measurements, the vertical photodetachment energies of deprotonated phenol and indole anions have also been recorded and presented. Quantum chemistry calculations support our assignments. We discuss our results in the context of the isolated protein chromophore anions acting as electron donors, one of their potential biological functions.

Using a combination of photoelectron spectroscopy measurements and quantum chemistry calculations, we have identified competing electron emission processes that contribute to the 350-315 nm photoelectron spectra of the deprotonated green fluorescent protein chromophore anion, phydroxybenzylidene-2,3-dimethylimidazolinone. As well as direct electron detachment from S 0 , we observe resonant excitation of the 2 1 ππ* state of the anion followed by autodetachment. The experimental photoelectron spectra are found to be significantly broader than photoelectron spectrum calculated using the Franck-Condon method and we attribute this to rapid (∼10 fs) vibrational decoherence, or intramolecular vibrational energy redistribution, within the neutral radical.

To understand how photoactive proteins function, it is necessary to understand the photoresponse of the chromophore. Photoactive yellow protein (PYP) is a prototypical signalling protein. Blue light triggers trans-cis isomerisation of the chromophore covalently bound within PYP as the first step in a photocycle that results in the host bacterium moving away from potentially harmful light. At higher energies, photoabsorption has the potential to create radicals and free electrons; however, this process is largely unexplored. Here, we use photoelectron spectroscopy and quantum chemistry calculations to show that the molecular structure and conformation of the isolated PYP chromophore can be exploited to control the competition between trans-cis isomerisation and radical formation. We also find evidence to suggest that one of the roles of the protein is to impede radical formation in PYP by preventing torsional motion in the electronic ground state of the chromophore.In nature, light drives many important processes such as photosynthesis, vision and phototaxis. At the heart of all these processes is a small chromophore whose photochemical response initiaties large-scale conformational changes in the protein wrapped around it, which in turn leads to a response at the cellular level. In addition to the desired photochemical reaction pathway, molecular chromophores are subject to competing processes such as internal conversion (IC), intersystem crossing and intramolecular vibrational redistribution and, in photoactive proteins in which the chromophore exists in a deprotonated anionic form, electron emission. [1][2][3][4] The role of the protein in controlling the competition between these process is still not understood fully.PYP is the primary photoreceptor for the negative photoactic response of the Halorhodospira * Present address: Physical and Theoretical