Unified Role of the 145th Residue on the Fluorescence\nLifetime of Fluorescent Proteins from the Jellyfish Aequorea victoria

Finding\na unified fluorescence mechanism is essential to develop\nand utilize fluorescent proteins appropriately. Here, we report the\nunified role of the 145th residue on the fluorescence efficiency of\nfluorescent proteins developed from the jellyfish Aequorea\nvictoria by demonstrating the difference and similarity\nbetween two representative fluorescent proteins, enhanced green fluorescent\nprotein (eGFP), and enhanced yellow fluorescent protein (eYFP). We\ndetermined the fluorescence lifetimes of the 19 different Y145 mutants\nof eGFP and eYFP by picosecond time-resolved fluorescence spectroscopy.\nWe found that the effect of the 145th mutation on the fluorescence\nlifetime is significant for eYFP but moderate for eGFP. We compared\nknown crystal structures to clarify the observed difference between\neGFP and eYFP. As a result, we conclude that the efficiency of the\nsteric restriction of the chromophore motion by the 145th side chain\nis essentially the same for both eGFP and eYFP. Meanwhile, the restriction\nof the chromophore motion by hydrogen bonds is more pronounced for\neGFP than for YFP. Balance of the steric effect and hydrogen bonding\ncontrols the lifetime of the Y145 mutants for eGFP and eYFP. Furthermore,\nthe steric restriction is induced by the electrostatic effect; the\ndifferent 145th residue induces a different electrostatic environment\naround the chromophore. The finding in this study reasonably explains\nthe reported lifetimes of other fluorescent proteins and allows the\nprediction of the lifetime of unknown fluorescent proteins from jellyfish.