Photophysics of the chromophore of the green fluorescent protein
Synthetic analogues of chromophores of green fluorescent protein were
synthesised. The chromophore, which is highly fluorescent in the protein, is nearly
non-fluorescent in solution. The spectral properties of the. model chromophores
(absorption coefficients, oscillator strengths and effect of pH), were compared with
those of the corresponding proteins. The measurements compared well with the
results of theoretical calculations. Some important differences between chromophore
and protein spectra were noted. The effects of solvent polarity on absorption and
emission spectra were studied and analysed using the rc* and the reaction field factor
measures of solvent effects. The spectral shifts were small and more dependent on
hydrogen bonding interactions than solvent polarity. The low temperature excitation
and emission spectra were recorded and compared to room temperature spectra. The
effect of excitation wavelength on the emission spectra was investigated at low
temperature and revealed an edge excitation red shift.
The spectral properties of the model chromophores were investigated as a
function of temperature. The fluorescence increased dramatically as the temperature
decreased, even into the supercooled liquid and glass phases. This suggests that the
mechanism of radiationless decay is not strongly coupled to solvent viscosity, but
may be thermally activated. An isoviscosity analysis to separate these two
mechanisms gave inconclusive results. The ultrafast polarisation spectroscopy of the
model chromophores was then investigated as a function of protonation state,
viscosity and temperature. The ground state recovery time has two components. An
ultrafast recovery time of <5ps is assigned to internal conversion followed by
vibrational relaxation. A slower time (>50ps) was assigned to rotational relaxation.
A second isoviscosity analysis suggested that the mechanism of radiationless decay
above room temperature was nearly barrierless, and only weakly coupled to viscosity.
These data are most consistent with internal conversion due to cooperative
intramolecular rotation of the chromophore.