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Fig. 11 Photoinduced
backbond cleavage and
b -elimination in “green-
to-red” photoconvertable
fluorescent proteins.
Absorbance spectra of green
and red forms of KikGRX
(a) and Kaede (b) after
denaturation (reproduced
with permission [ 71 ])
His62 in EosFP, followed by a
-elimination step in which Glu212 acts as a proton
acceptor [ 66 ]. Glu212 is known to be essential for photoconversion of EosFP, since
the E212Q mutant does not photoconvert [ 66 ]. Hayashi et al. [ 73 ] suggested a
“water-assisted” mechanism to explain the loss of a water molecule in the red
photoproduct of Kaede [ 73 ]. Tsutsui et al. [ 71 ] discuss one specific detail of the
-elimination, which can follow either a E2-type elimination which is bimolecular,
or an E1-type elimination which is unimolecular. Interestingly, the red photoproduct
in KikGR(X) was found to be structurally different compared to Kaede and EosFP,
having the C
in a cis rather than a
trans configuration [ 71 ]. This shows that free bond rotation occurs in a reaction
intermediate that favours the “E1” mechanism of
C double bond between His62-C
and His62-C
Lelimousin et al. [ 74 ] proposed that intersystem crossing occurs in the reaction
pathways, arguing that this explains the low quantum yield of photoconversion
[ 74 ]. Hybrid QM/MM calculations were used to calculate the relative energies of
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