Primary Photophysical Processes in Chromoproteins - Fluorescent Proteins: From Understanding to Design - page 52

and location of substituents, as might have been predicted from the calculation [ 58 ].
However, in all cases the fluorescence quantum yield remains low.
The mechanism by which the protein renders the chromophore fluorescent has still
not been definitively determined. Many calculations suggest that an almost 90 rotation
about one or other of the bridging bonds is required for the closest approach of ground
and excited states. It is easy to imagine that such a large volume excited state
reorganisation could be sterically hindered by the protein, and molecular mechanics
calculations suggest that many possible coordinates are so restricted [ 86 , 87 ]. However,
experiments in viscous media suggest that the coordinate promoting IC is volume
conserving or at least has a large driving force, neither of which are in agreement with
calculated coordinates requiring a large excited state reorganisation. Calculations do,
however, suggest that the excited state relaxation pathway is sensitive to the chromo-
phore's environment. It is also likely (e.g., on the basis of spectroscopic studies, section
3.1) that the electronic structure of the chromophore is modified on incorporation in the
protein environment. It seems probable that some combination of steric and chemical
(e.g., H-bond) effects can suppress excited state structure change, while environmental
effects (charges on nearby residues, pi stacking, H-bonds) can modify the driving
force, and that these factors operating together suppress IC. However, there is as yet
no predictive model of the CP fluorescence enhancement mechanism.
While the mechanism suppressing IC remains to be determined, there is struc-
tural evidence to suggest that (at least some) CPs that are essentially non-fluorescent
have a chromophore that exists in a non-planar ground state conformation [ 88 , 89 ].
Conversely, it has been noted that both non-fluorescent and (weakly) fluorescent
forms of a coral protein exhibit non-planar chromophore structures [ 90 ]. This
suggests that more than one feature of the chromophore in the protein is responsible
for controlling the fluorescence yield. Recently, a sterically crowded HBDI deriva-
tive formed by locating methyl groups in positions meta to the phenolic hydroxyl
was studied. DFT calculations reveal a strongly twisted ground state. This derivative
indeed exhibits extremely rapid fluorescence decay (ca. 100 fs even in the anionic
form) [ 58 ]. This is consistent with the proposed correlation between non-planar
ground state and fast radiationless decay. However, the analogy between synthetic
chromophore and CP is not exact, as the non-planar derivative has a blue-shifted
absorption relative to HBDI, while the non-planar non-emissive CPs are usually red
absorbing.
4 ESPT in avGFP and Its Mutants
In the preceding section, it was shown that the photophysics of the GFP chromo-
phore in solution are dominated by ultrafast structural changes resulting in IC.
The effect of the protein matrix is so dramatic that IC can be neglected in
avGFP, and the photophysics are instead dominated by a completely different
mechanism, ESPT. Excitation into either neutral (usually labelled the A state) or
anionic (B state) absorption bands of avGFP results in an intense green emission