Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 202

Fig. 15 Proposed mechanism of the reversible photoswitching mechanism of asFP595, repro-
duced with permission from Schafer et al. [ 63 ]. QM/MM simulations have led to the proposal that
protonation controls the photoswitching in asFP595. Legend from original figure: “The fluorescent
state Zcis is highlighted . The green arrows indicate ground state equilibria, whereas the red
arrows indicate excited state processes. The major protonation states are the zwitterionic and
the anionic chromophores in the trans conformation, and the neutral chromophore in the cis
conformation, as indicated in the square bracket ”
In addition, the timescale of the deactivation 320 fs [ 98 ] would be very fast for the
proposed ESPT reaction [ 63 ]. A number of theoretical calculations evaluated the
potential energy of excited states of GFP chromophore models in the different proton-
ation states [ 96 , 99 ]. These suggest that the zwitterions have a conical intersection in a
twisted geometry involving the rotation of only a single bond [ 96 ].
Li et al. [ 90 ] performed QM/MM calculations to study the photoswitching in the
related Dronpa protein [ 90 ]. In contrast to the results for asFP595 [ 63 ], these
calculations supported the anionic cis form for the “on” state and the neutral
trans conformation for the “off” state of Dronpa [ 90 ]. The protonation is mentioned
to be of critical importance for the non-fluorescent (dark) state of the trans -oriented
chromophores in both asFP595 and Dronpa. It would appear that the general trend
is that cis chromophores are bright states and trans chromophores are dark states.
However, an exception is the eqFP611 fluorescent protein from A. sulcata which
has a trans -oriented Met-Tyr-Gly-derived chromophore with a relatively high
fluorescence quantum yield of 0.45 [ 103 , 104 ]. It is possible that in the trans
orientation of the eqFP611, chromophore is not in the zwitter-ionic state, which
would support the conclusions reached for asFP595 [ 63 , 64 ]. Vibrational spectros-
copy information is not yet available to address this issue.