Structure–Function Relationships in Fluorescent Marker Proteins of the Green Fluorescent Protein Family - Fluorescent Proteins: From Understanding to Design - page 247

Photoactivated Modifications of the Chromophore

In addition to the spontaneous chemical alterations of the green chromophore
described above, strictly photoinduced modifications can occur in a variety of
FPs. The first photoactivatable (PA) FP - PA-GFP - was derived from wild-type
GFP by a single point mutation, Thr203His [ 10 ]. This variant is devoid of fluores-
cence until activated by irradiation with intense violet light. X-ray structures of PA-
GFP determined before and after irradiation showed that photoactivation is the
result of a light-induced decarboxylation of the Glu222 side chain that shifts the
chromophore equilibrium from the neutral to the anionic form [ 79 ], which shows
bright green fluorescence. Because the mutant is not visible before light irradiation,
it is difficult to identify suitable regions to be photoactivated in a biological sample,
which limits the applicability of this protein.

Irreversible Photoconversion from Green to Red Emission

In another class of photoconvertible FPs that includes Kaede [ 80 ], EosFP [ 52 , 81 ],
and a few other proteins [ 67 , 82 - 84 ], irradiation into the absorption band of the
neutral green chromophore (~400 nm) shifts the emission maximum from the green
to the red. This process is irreversible and associated with a backbone cleavage
between the N

atoms of the first amino acid in the chromophore-forming
tripeptide, which is always a histidine in this class (Fig. 6 ). Concomitantly, a double
bond forms between its C

a and C a

-conjugation is
extended via an all-trans ethenylene moiety into the histidine imidazole ring.
Similar to DsRed and AsRed, the protein emits orange-colored fluorescence at
~580 nm after photoconversion, suggesting that the increased delocalization is
mainly due to the double bond extension by the ethenylene.
The exact mechanism governing photoconversion is still unclear, although
several hypotheses have been proposed. Mizuno et al. suggested initially that
the imidazole of His62 could become biprotonated and thus facilitate backbone
cleavage via

a and C b atoms, so that the p -HBI p

b -elimination, accompanied by an extension of the conjugated

Fig. 6 Backbone cleavage upon green-to-red photoconversion of EosFP. Left : green EosFP.
Right : red EosFP