Stable low-temperature photoproducts and hole burning of green fluorescentprotein (GFP)

We present low-temperature optical investigations down to 2 K of the photoc hemistry 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 fluores cence spectroscopy. The data suggest that P502 is identical with the obliga tory intermediate of the excited-state deprotonation cycle R-neq(-) that ex ists only as a transient at room temperature. The second new product P510 i s also associated with R-neq(-), whereas P489 is related to the equilibrate d 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-temper ature persistent spectral hole burning in the red wing of the R-eq(-) absor ption has been demonstrated and can be used to characterize electron-phonon coupling.