Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 199

this state is, however, not yet fully understood in detail. Similar behaviour was
reported for the photoactivatable red fluorescent protein PA-mRFP1s [ 62 ].

5 Reversible Photoconversion Reactions Resulting from
Chromophore Photoisomerisation in Fluorescent Proteins

Yet another distinct class of photoconvertable fluorescent proteins concerns mutants
of avGFP and natural and modified GFP homologues which undergo photoisome-
risation reactions. These reactions are generally both thermally and photochemically
reversible, and significant changes in the fluorescence quantum yield allows their
use in protein-tracking applications in fluorescence microscopy. Additionally, the
quantum yield of photoisomerisation can exceed that of any of the other photocon-
version mechanisms that are known to date.
This class of proteins is called “reversibly switchable fluorescent proteins”
(RSFPs). Examples displaying reversible photoconversion between dark and bright
state are Dronpa [ 79 - 81 ] and its fast-switchable variant rsFastLime [ 81 ], asFP595
(asulCP, asCP) [ 82 - 85 ] and the kindling fluorescent protein (KFP; the A148G
mutant of asFP595) [ 60 ], Padron [ 86 ] and IrisFP [ 87 ].It is established that cis - trans
isomerisation of the chromophore is involved in switching, but protonation changes
of the chromophore have also been implicated [ 63 , 64 , 88 - 90 ]. The role of photo-
isomerisation in RSFPs is currently well established, but recent studies, most of
them computational, disagree on the protonation state and possible proton transfer
reactions in the photoconversion reactions.
The “Dronpa” protein from the coral Pectiniidae undergoes photoswitching in
the crystalline state. Andresen et al. [ 80 ], Stiel et al. [ 81 ] and Mizuno et al. [ 91 ]
performed X-ray diffraction experiments with photoswitchable Dronpa crystals.
Both in solution and in crystals, the dark resting state has a high fluorescence
quantum yield of 0.85, with absorption and emission maxima at 503 and 522 nm
[ 81 ]. Illumination with 500 nm light photoconverts the ground state to a non-
fluorescent (fluorescence quantum yield of 0.02) protonated species absorbing at
380 nm, which can be re-transformed with illumination at ~400 nm [ 79 ]. The crystal
structure of the fluorescent state showed the Cys-Tyr-Gly-derived chromophore to
be in a cis conformation[ 81 ], as the avGFP chromophore [ 4 , 6 ]. Subsequent illumi-
nation with 488 nm light switches the protein to a non-fluorescent state with an
absorption maximum at 380 nm with a quantum yield of 3.2 10 4 [ 92 ] which

decays back to ground state with an 18-h time constant [ 81 ]. Several studies found
that the crystal structure of the photoproduct had the chromophore in a trans
configuration, in addition to structural changes in the chromophore-binding site,
possibly leading to stable protonation of the chromophore phenolic oxygen [ 80 , 89 ,
91 ]. The protonation in the trans off-state is inferred from electrostatics calculations
of the chromophore environment and the wavelength of electronic absorbance [ 80 ],
but this currently needs to be confirmed from FTIR spectroscopy and/or pH titra-
tions. Subsequent illumination of the trans off-state with ~400 nm light causes very