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The possibility of the ESPT in asFP595 initiated from the imidazolidinone
nitrogen [
63
] is unusual and suggests that the protein environment would have
pronounced effects on the photophysical properties of the chromophore, since
ESPT in photoacids is typically found to be initiated from a phenolate group. In
the latter picture, the imidazolidinone nitrogen could act as a photobase rather than
a photoacid [
102
]. Critical in the interpretation is the assignment of the transitions to
the electronic species. From the QM/MM calculations, Schafer et al. [
64
] assigned
the low energy transition to the zwitter-ion in both
cis
and
trans
forms of asFP595
[
64
]. In contrast, Bravaya et al. [
105
] assign the low energy transition to the anionic
state and predict the zwitter-ion at higher energy [
105
]. The calculations indicated
that the S0-S1 transition energy for the zwitter-ion in the gas phase is particularly
sensitive to the level of theory used and showed that multireference perturbation
theory calculations [
105
] do not agree with TD-DFT [
105
] or ZINDO [
64
] results.
Taking a different approach, Olsen et al. [
95
] performed quantum mechanical
calculations, which indicated that photoisomerisation occurs at different locations
depending on the protonation state of the chromophore [
95
]. Specifically the calcu-
lations indicated excited state torsion of the phenoxy bridge in the neutral and the
imidazolidinone bridge dihedral angles for the anionic chromophore [
95
].
The quantum yield of the
cis
on to
trans
off photoswitching is much lower,
although mutations have led to increased rates of photoconversion [
81
,
106
]. The
possibility of intersystem crossing in the
cys
to
trans
photoisomerisation has been
discussed [
91
,
92
]. The involvement of a triplet state is a distinct possibility
also in this photoconversion reaction, and has also previously been entertained in
other types of (irreversible) photoconversion reactions (see Sects.
3
and
4
). Because
of the low quantum yield, spectroscopic studies of the
cis
on to
trans
off photo-
switching are more challenging. In addition, the rate constant for thermal reversion
differs substantially in different fluorescent proteins, from 7s in asFP595 and 50s
in the A148G mutant of asFP595 (KFP) [
60
].
From the multitude of photoconversion reactions, there also exist fluorescent
proteins that display more than one type. One example is IrisFP that undergoes both
cis
-
trans
photoisomerisation reactions as well as irreversible “green-to-red” photo-
conversion as found in “EosFP” (which IrisFP is derived of by mutagenesis) and
related proteins (Sect.
4
)[
87
].
6 General Conclusions
GFP and related fluorescent proteins not only provide a diverse palette of colours,
but also display a very broad range of different types of photoreactions that can be
exploited in fluorescence microscopy applications. These include irreversible and
reversible photoconversion reactions that are associated with spectroscopic changes
resulting in significant contrast. The photoconversion of GFP, which is the primary
focus of this chapter, has been reviewed. While the principal molecular and
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