Photochromicity and fluorescence lifetimes of green fluorescent protein

The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequore a and its mutants have gained widespread usage as an indicator of structure and function within cells. Proton transfer has been implicated in the comp lex photophysics of the wild-type molecule, exhibiting a protonated A speci es excited at 400 nm, and two deprotonated excited-state species I* and B* with red-shifted excitation similar to 475 nm. Photochromicity between the protonated and deprotonated species has been reported upon 400 nm excitatio n. Using precise time-resolved spectroscopy, we have been able to distingui sh the fluorescence lifetimes of the I and B species (similar to 3.3 and si milar to 2.8 ns, respectively) and show that the irreversible photochromici ty which we observe is due to formation in the excited state of the B speci es, which cannot return to other species in the ground state. The ground st ate A and I species are in thermal equilibrium. Anistropy measurements indi cate chat the chromophore lies rigidly in the molecule with a rotational co rrelation time of similar to 15.5 ns, as is to be expected for a molecule o f this size. Time-resolved measurements of enhanced yellow fluorescent prot ein (EYFP) and red-shifted green fluorescent protein (RSGFP) were also anal yzed.