Origin of the Intrinsic Fluorescence of the Green Fluorescent Protein

Green fluorescent protein, GFP, has revolutionized biology, due to, its use in bioimaging. It is widely accepted that the protein environment makes its chromophore fluoresce, whereas the fluorescence is completely lost when the, native chromophore is taken out of GFP. By the use of a new femtosecond pump-probe scheme, based on time-resolved action spectroscopy, we demonstrate that the isolated deprotonated GFP chromophore can be trapped in the first excited state when cooled to 100 K. The trapping is Shown to last fot 1.2 ns, which is long enough to establish conditions for fluorescence and consistent with calculated trapping barriers in the electronically excited state: Thus, GFP fluorescence is traced back to an intrinsic chromophore property, and by improving excited-state trapping protein interactions enhance the molecular fluorescence.