The photophysics of fluorescent protein chromophores
Fluorescent proteins of the Green Fluorescent Protein (GFP) family are important and versatile scientific tools used in a range of applications including the labelling of proteins, cells and tissue, high resolution imaging and studying protein-protein interactions. They exhibit a very diverse range of photochemical behaviour. Surprisingly this diversity originates from chromophores which have very similar structures. Accordingly, minor changes to the chromophore structure or to its surroundings in the protein can produce dramatic changes in optical behaviour. In this work the photophysics of the chromophores of the green fluorescent protein, the Kaede fluorescent protein and some related analogues are investigated using ultrafast methods; predominantly the excited state is probed using ultrafast fluorescence up-conversion with a time resolution of better than 50 fs. The excited states of chromophores decay on the ultrafast timescale. The decay will be probed for both isomers of the GFP chromophore, and the mechanism of decay will be modelled, leading to conclusions on the nature of the radiationless coordinate. The derivative of the chromophore generated in the Kaede protein will be studied, and marked differences with the GFP chromophore found. The importance of protein-chromophore interactions in influencing fluorescent protein behaviour will be demonstrated. The effects of chromophore modifications will be revealed through comparison of the analogues and the sensitivity of chromophore photophysics to the hydrogen bonding nature of the solvent will be demonstrated. It will also be shown that the Kaede chromophore undergoes aggregation enhanced fluorescence at relatively low concentrations.
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