Photoconversion of the Green Fluorescent Protein and Related Proteins - Fluorescent Proteins: From Understanding to Design - page 198

the predicted intermediates, involving ICS from S1 to T1 and finally to S0 [ 74 ].
These calculations have attempted to distinguish between the reaction models
proposed by Nienhaus et al. [ 66 ] and Mizuno et al. [ 72 ] by comparing the energy
minimised reaction trajectories. Interestingly, it was argued that the involvement of
a triplet state may show that ESPT would not participate in the photoconversion
reaction [ 74 ]. Awaiting ultrafast spectroscopy and cryo-trapping studies with this
class of photoconvertable proteins there is currently no consensus in the literature
with regard to the electronic configuration of reaction intermediates.

4 Light-Induced Maturation in Related Fluorescent Proteins

Oxidative decarboxylation of Glu222 was initially proposed to occur in a new type of
UV-induced photoconversion that was reported for a mutant of a GFP homologue,
a non-fluorescent jellyfish A. coerulescens chromoprotein which caries the same SYG
motif for the chromophore, called “aceGFPL” [ 75 ]. Random mutagenesis identified
an E222G mutation which leads to a fluorescent derivative, called “aceGFP”. The
GFP-like wild-type protein, or rather the “back-mutated” aceGFP-G222E variant,
when expressed in E. coli is colourless and has only very minor absorption at 390 nm
indicating a minor neutral chromophore species. Illumination with UV light in the
250-300 nm region resulted in photoconversion, generating an anionic species, with
480nmexcitationand505nmemissionatroomtemperature[ 75 ]. Although further
characterisation was not performed, it was suggested that light-induced electron
transfer leading to decarboxylation of Glu222 could be part of the photochromic
reaction. It appears, however, that UV light-induced photoconversion triggers matu-
ration of the anionic chromophore specifically, whereas the minor neutral species does
not convert. Recently the crystal structure of aceGFP-G222E was presented, which
showed that the chromophore was not matured with C atoms in the sp3

hybridisation [ 76 ]. The crystal structure of the photoconverted aceGFP-G222E, which
has the anionic chromophore, did not indicate oxidative decarboxylation of Glu222.
The observation is discussed in the light of the different proposals for chromophore
maturation mechanisms [ 77 , 78 ], particularly supporting a dehydrated, cyclised
chromophore structure in the immature state in the aceGFP- G222E protein, as
proposed by [ 77 ]. The photochemistry of UV-induced maturation starting with

a and C b

Fig. 12 (continued) Gly64 are drawn; the neighbouring amino acids (single-letter code) are also
shown. (a) The green chromophore in the initial protonated state. (b) Excitation of the chromo-
phore by ultraviolet light. (c) Excited-state proton transfer. (d) The excited ionized phenolic
hydroxyl group and protonated imidazole ring of His62. (e) Resonance stabilization of the
intermediate structure. Allowed rotation of the imidazole ring along the bond between His62-C a
and His62-C b is indicated by arrows . The leaving group is carboxamidic acid, which becomes
carboxamide through tautomerization. (F and H) Tautomerization with (H) and without (F)
rotation of the imidazole ring (G and I). Loss of a proton from C b leads to completion of the

red chromophore with either a trans (G) or cis (I) double bond”