Photochromicity and Fluorescence Lifetimes of Green Fluorescent Protein

The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the complex photophysics of the wild-type molecule, exhibiting a protonated A species excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation ∼475 nm. Photochromicity between the protonated and deprotonated species has been reported upon 400 nm excitation. Using precise time-resolved spectroscopy, we have been able to distinguish the fluorescence lifetimes of the I and B species (∼3.3 and ∼2.8 ns, respectively) and show that the irreversible photochromicity which we observe is due to formation in the excited state of the B species, which cannot return to other species in the ground state. The ground state A and I species are in thermal equilibrium. Anisotropy measurements indicate that the chromophore lies rigidly in the molecule with a rotational correlation time of ∼15.5 ns, as is to be expected for a molecule of this size. Time-resolved measurements of enhanced yellow fluorescent protein (EYFP) and red-shifted green fluorescent protein (RSGFP) were also analyzed.