Stable Low-Temperature Photoproducts and Hole Burning of Green Fluorescent Protein (GFP)

We present low-temperature optical investigations down to 2 K of the photochemistry and hole burning of wild-type green fluorescent protein (wt-GFP). Three new photoproducts with absorption at 489, 502, and 510 nm (P489, P502, P510, respectively) are identified by fluorescence excitation and fluorescence spectroscopy. The data suggest that P502 is identical with the obligatory intermediate of the excited-state deprotonation cycle R-neq that exists only as a transient at room temperature. The second new product P510 is also associated with R-neq, whereas P489 is related to the equilibrated anionic form R-eq. An extended energy scheme accounting for these new species is presented. The ground-state energy barriers characterizing these products have been determined by temperature-dependent studies. Low-temperature persistent spectral hole burning in the red wing of the R-eq absorption has been demonstrated and can be used to characterize electron−phonon coupling.