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Fig. 14 Comparison of the
on-state ( blue ) and off-state
( green ) structures of Dronpa
in the chromophore vicinity
(reproduced with permission
from Andresen et al. [ 80 ])
Dronpa, photoswitching of asFP595 also involves protonation change of the chromo-
phore [ 94 ]. However, in asFP595, the imidazolidinone nitrogen is proposed to form
a zwitter-ionic species, on the basis of theoretical work [ 63 , 64 ](Fig. 15 ).
Several computational and simulation studies have focused on the photoswitch-
ing dynamics of “RSFPs” [ 63 , 64 , 90 , 95 , 96 ]. Sch
afer et al. [ 63 , 64 ] proposed that
the chromophore is present as a zwitter-ion in the trans form, and that ESPT
proceeds from the imidazolidinone nitrogen (rather than the phenolic oxygen). In
GFP, the zwitter-ion was shown not to be present. For the free chromophore model
compound ethyl 4-(4-hydroxyphenyl)methylidene-2-methyl-5-oxoimidazolacetate,
Bell et al. [ 97 ] determined a pKa value of 8.0 for protonation of the imidazolidinone
nitrogen [ 97 ] and showed from resonance Raman spectroscopy that in GFP at pH
8.0, where a mixture of anionic and neutral species is present, the position of the
Raman bands exclude the possibility of cationic and zwitter-ionic forms [ 97 ]. In the
case of asFP595, an acidic carboxylate from Glu215 is in hydrogen-bonding contact
with the imidazolidinone nitrogen [ 82 , 84 , 85 ]. QM/MM simulations hitherto
suggest that the zwitter-ion is the dominant species [ 63 , 64 ], but this is yet to be
confirmed by vibrational spectroscopy. Schafer et al. [ 63 ] propose that ESPT
deactivates the otherwise fluorescent zwitter-ionic trans chromophore, explaining
the low fluorescence quantum yield [ 63 , 64 ]. Indeed, ultrafast spectroscopy was
used to determine the lifetime of the excited state, and found a dominant decay
component of 320 fs, corresponding to the low fluorescence quantum yield < 10 4
[ 98 ]. The existence of a dark zwitter-ionic state was proposed early on from semi-
empirical calculations of the GFP chromophore [ 99 ], providing a direct explanation
for “blinking” behaviour observed at the single molecule level [ 100 , 101 ]. Semi-
empirical calculations predicted that in GFP in the ground state the zwitter-ion is not
populated [ 102 ].Furthermore, the calculated excited-state free energy levels show
the expected increased acidity of the phenol oxygen and increased basicity of the
imino nitrogen [ 102 ]. This agrees with a picture of charge migration in the excited
state typical of photoacid behaviour, and counters the results from Sch
afer et al.
[ 63 , 64 ] who propose deactivation of the excited zwitter-ion state via proton transfer.
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