Excitation wavelength dependence of the proton-transfer reaction of the green fluorescent protein

Picosecond time-correlated single-photon counting was used to measure the proton-transfer rate of green fluorescent protein (GFP) excited by several wavelengths between 266 and 405 nm. When samples of GFP in water and D ₂O are excited at short wavelengths, λ_ex < 295 nm, the fluorescence properties are largely modified with respect to excitation at a wavelength around 400 nm, the peak of the absorption band of the S ₀ → S₁ transition of the ROH form of the chromophore. The shorter the excitation wavelength, the longer the decay time of the ROH emission band at 450 nm and the longer the rise time of the RO^- emission band at 512 nm. The proton transfer is slower by an order of magnitude and about a factor of 3 when GFP in water and D₂O are excited by 266 nm, respectively.