Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 194

decarboxylation could also be at Glu222, as in the wild type. The fact that only
pulsed excitation causes decarboxylation would suggest that light-induced electron
transfer in YFP is triggered by a multi-photon process.
Another A. victoria GFP mutant has been reported to undergo classical GFP A !
GFP R photoconversion involving decarboxylation. A T203H mutation shows no
ground state population of the minor anionic species that is observed in the wild
type and only shows the GFP A neutral species absorbing at 398 nm [ 35 ], which is
rationalised on the basis of an altered orientation of the Glu222 carboxylate [ 54 ].
Therefore, the spectroscopic contrast with photoconversion is enhanced when the
anionic form is monitored. This improved contrast has obvious utility for fluorescence
microscopy applications [ 35 ]. The mutant is referred to as “photoactivatable GFP”
(PA-GFP) and includes secondary mutations F99S/M153T/V163A that do not appear
to affect the spectroscopic properties [ 34 , 35 , 55 , 56 ]. With regard to the photoconver-
sion mechanism, it was shown to be the same as that of the wild type [ 54 ].
The Aequorea coerulescens aceGFP mutant “photoswitchable cyan fluorescent
protein” (PS-CFP) was developed as an alternative to “PA-GFP” for protein
tracking applications [ 57 - 59 ]. Photoconversion switches the fluorescence extinc-
tion and emission maxima from 402 and 468 nm to 490 and 511 nm, similar to PA-
GFP. However, the fluorescence quantum yield of the neutral and anionic species is
0.16 and 0.19, respectively, and similar extinction coefficients are reported for both.
While PA-GFP is significantly brighter, the difference of the fluorescence emission
of neutral and anionic species can lead to improved contrast in emission [ 57 , 59 ].
Although not fully characterised, light-induced electron transfer and oxidative
decarboxylation is proposed to occur in PS-CFP as in PA-GFP and wild-type
GFP. [ 57 - 59 ]. Although an estimate of the quantum yield is not available, conver-
sion was reported to occur efficiently with 10 ns pulses of 15

J energy at 404 nm
with no spot size reported [ 59 ]. Therefore, the photoconversion quantum yield of
PS-CFP could possibly be higher than that for GFP at 400 nm.
The fluorescent protein DsRed from the coral Discosoma sp. undergoes photo-
conversion of the red ground state (R) absorbing at 559 nm and emitting at 583 nm
with a pulsed laser at 532 nm into a red-shifted super red (SR) species, absorbing at
574 nm and emitting at 595 nm. The fluorescence quantum yield of the photoprod-
uct is 0.012, in contrast to the bright R state which has a fluorescence quantum yield
of 0.7. Photoconversion of DsRed is complex and thought to involve cis - trans
isomerisation as well as oxidative decarboxylation, proposed to be of Glu215, the
equivalent of avGFP Glu222. In addition, a minor photoproduct that has a proto-
nated chromophore is observed [ 40 ]. Mass spectrometry and FTIR spectroscopy
suggest the oxidative decarboxylation of a carboxylic acid in the anionic form from
the loss of bands of 1,568 and 1,389 cm 1 , proposed to belong to the COO
stretching modes [ 40 ], similar to those observed in GFP [ 20 ]. The proposed oxida-
tive decarboxylation likely follows the same mechanism as in GFP, via the Kolbe
mechanism [ 19 ]. A complex and interesting mechanism is proposed for the photo-
conversion of DsRed. The cavity created after decarboxylation of the nearby
Glu215 is proposed to allow sufficient motion for cis - trans isomerisation of the
DsRed chromophore to occur [ 40 ]. Nevertheless, in photoconverted samples, the

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